VEGFR Fusion Protein Pharmaceutical Composition

ABSTRACT

The present invention relates to a biologic that inhibits angiogenesis. In particular, the present invention relates to fusion proteins that inhibit the integrin activated pathway and one other angiogenic factor-activated pathway as well as formulation compositions of such fusion proteins, as well as methods for producing and using the same.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation application of U.S. Non-Provisionalpatent application Ser. No. 17/663,260, filed May 13, 2022, thedisclosure of which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The present invention relates to a fusion protein pharmaceuticalcomposition that inhibits angiogenic factor-activated pathways. Inparticular, the present invention relates to fusion proteins thatinhibit angiogenic factor-activated pathways, the compositions of thesefusion proteins, as well as methods for producing and using the same.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The contents of the electronic sequence listing(SequenceListing7US2.xml; Size: 91,700 bytes; and Date of Creation: Mar.15, 2023) is herein incorporated by reference in its entirety.

BACKGROUND

Angiogenesis is the process of growing new blood vessels from theexisting vasculature. It plays an important role in severalphysiological processes, including embryonic development, as well astissue and wound repair (Folkman J et al. Angiogenic Factors. Science1987; 235:442-7). The physiologic steps of angiogenesis are wellcharacterized, and involve proteolysis of the extracellular matrix,proliferation, adhesion, migration, and assembly of the endothelialcells into a tubular channel, mural cell, pericyte recruitment anddifferentiation, and extracellular matrix production (Carmeliet P et al.Nature. 2011; 473:298-307). Pathologic angiogenesis may occur in tumorformation, ocular disorders (e.g., diabetic retinopathy, diabeticmacular edema, retinal/choroidal neovascularization, exudativeage-related macular degeneration, and neovascular glaucoma), arthritis,psoriasis, fibrotic diseases, inflammatory diseases, atherosclerosis,and arteriosclerosis (Polverini P J. Crit Rev Oral Biol Med. 1995;6(3):230-47, Perrotta P et al. Vascular Pharmacology. 2019; 112:72-78).

Pathologic angiogenesis is more heterogeneous and chaotic, oftendemonstrating tortuous vessel organization, hypoxic voids of varioussizes, uneven and imperfect vessel walls and linings, and ineffectiveperfusion (Jain R K., Nat Med. 2003; 9(6):685-93). These distinctcharacteristics of new blood vessel formation in diseases have madetherapeutic targeting of angiogenesis a challenge. Although anti-VEGFtherapies such as LUCENTIS® (ranibizumab), EYLEA® (aflibercept), oroff-label use of AVASTIN® (bevacizumab) can generally stabilize orimprove visual function, sub-retinal scarring (fibrosis) can develop inapproximately half of all treated eyes within two years after anti-VEGFtreatment and has been identified as one cause of unsuccessful outcomes(Daniel E et al. Ophthalmology. 2014; 121(3):656-66). Many of thecritical players in sub-retinal fibrosis are likely to be the growthfactors and the matricellular proteins that are involved in the fibroticprocess (cell proliferation, migration and ECM remodeling) (Patsenker Eet al. Hepatology. 2009 November; 50(5): 1501-1511, Xu J et al. BiochimBiophys Acta. 2014 November; 1842(11): 2106-2119). Despite itscomplexity, with our increasing knowledge of the angiogenic process,anti-angiogenic drug development remains an area of great interest.

Currently, many key players in the neovascularization process have beenidentified, and the vascular endothelial growth factor (VEGF) family hasa predominant role. The human VEGF family consists of 6 members: VEGF-A,VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PlGF). Inaddition, multiple isoforms of VEGF-A, VEGF-B, and PIGF are generatedthrough alternative RNA splicing (Sullivan et al. MAbs. 2002; 2(2):165-75). VEGF-A is the primary factor involved with angiogenesis; itbinds to both VEGFR-1 and VEGFR-2. The strategy of inhibitingangiogenesis by obstructing VEGF-A signaling has established successfultherapies for treatment of specific cancers as well as retinalneovascular and ischemic diseases. (Major et al. J Pharmacol Exp Ther.1997; 283(1):402-10; Willet et al. Nat. Med. 2004; 10:145-7;Papadopoulos et al. Angiogenesis. 2012; 15(2):171-85; Aiello et al.PNAS. 1995; 92:10457-61).

Other growth factors, cytokines, chemokines including Platelet DerivedGrowth Factors (PDGFs), Transforming Growth Factors beta (TGF-β),Epidermal Growth Factors (EGFs), Nerve Growth Factors (NGFs),Hypoxia-Induced Factor (HIF), basic Fibroblast Growth Factor orFibroblast Growth Factor (bFGF or FGF-2), Connective-Tissue GrowthFactor (CTGF), Granulocyte-Macrophage Colony-Stimulating Factor(GM-CSF), Insulin-Like Growth Factor (IGF), Hepatocyte GrowthFactors/Scatter Factor (HGF/SF), Tumor Necrosis Factor alpha (TNF-α),stromal cell-derived factor-1 (SDF-1), Interleukin 1 (IL-1), Interleukin6 (IL-6), Interleukin 8 (IL-8), Interleukin 17 (IL-17), Interleukin 18(IL-18), Interleukin 20 (IL-20), Interleukin 23 (IL-23),Chemoattractants such as C—C motif Ligand (CCL28, CCL21) and C—X—C motifLigand (CXCL1, CXCL5), Macrophage migration Inhibitory Factor (MIF), andimmune cell surface proteins such as Clusters of Differentiation (CDs).These factors are reported to be overexpressed and play key roles inangiogenesis-related diseases (Elshabrawy et al. Angiogenesis. 2015;18:433-448; Somanath P R et al, Cell Biochem Biophys. 2009; 53(2):53-64, Eliceiri B P., Circ Res. 2001 Dec. 7; 89(12):1104-10). Targetingthese factors to reduce their downstream pathway activation may decreaseangiogenesis-related diseases.

Integrins, a family of cell surface receptors, are also found to beoverexpressed on the endothelial cell surface and are believed tofacilitate the growth and survival of newly forming vessels duringangiogenesis. Integrins are heterodimeric cell surface receptors thatinteract with extracellular matrix proteins and are critical for manybiological processes. The expression of integrins in various cell typesare involved in tumor progression, and their ability to crosstalk withgrowth factor receptors and directly interact with several growthfactors has made them attractive therapeutic targets. (Staunton D E etal. Adv Immunol. 2006; 91:111-57; Avraamides, C J et al. Nat Rev Cancer.2008; 8:604-617, Somanath P R et al. Cell Biochem Biophys. 2009; 53(2):53-64) In particular, the integrin αvβ3 is upregulated in both tumorcells and angiogenic endothelial cells, and is important for tumor cellmigration, angiogenesis/neovascularization, and dysregulated cellsignaling. Therefore, antagonists of the integrin αvβ3 are intensivelystudied for their anti-angiogenic and anti-tumor properties(Desgrosellier J S et al. Nat Rev Cancer. 2010; 10:9-22).

Disintegrins are proteins found in snake venom of the viper family andmainly inhibit the function of β1- and β3-associated integrins. Theywere first identified as inhibitors of integrin αIIbβ3 and weresubsequently shown to bind with high affinity to other integrins,blocking the interaction of integrins with RGD-containing proteins. Theycontain 47 to 84 amino acids with about 4 to 7 disulfide bonds and carrythe same RGD motif (McLane M A, et al. Proc Soc Exp Biol Med. 1998; 219:109-119; Niewiarowski S et al. Semin Hematol 1994; 31: 289-300; CalveteJ J, Curr Pharm Des. 2005; 11: 829-835; Blobel C P et al. Curr Opin CellBiol. 1992; 4: 760-765). The conserved RGD sequence in the disintegrinfamily plays the most important role in recognizing the integrins.Disintegrins were found to interact with eight out of twenty-fourintegrins and inhibit integrin-mediated cell proliferation, adhesion,migration, and angiogenesis (McLane M A, et al. Front Biosci. 2008; 13:6617-6637; Swenson S, et al. Curr Pharm Des. 2007; 13: 2860-2871).Animal studies showed that disintegrins targeted neovascular endotheliumand metastatic tumors, indicating their potential use in cancer therapy.The specific binding of RGD-containing proteins to integrin is afunction of both the conformation and the local sequence surrounding theRGD motif. Many studies have shown that the residues flanking the RGDmotif of RGD-containing proteins affect their binding specificities andaffinities to integrins (Scarborough R M et al. J Biol Chem. 1993; 268:1058-1065; Rahman S et al. Biochem J. 1998; 335: 247-257).

Angiogenesis is a complex biological process which involves variousgrowth factors and signaling receptors and targeting single molecules inthe signaling cascade may not provide an effective clinical treatmentfor uncontrolled angiogenesis in diseases such as cancer. Therefore,there is a growing need to develop innovative therapeutics capable ofbinding several key angiogenic factors in a cooperative manner toeffectively inhibit angiogenesis and progression of the disease.

BRIEF SUMMARY

Provided herein are pharmaceutical formulations of fusion proteins andmethods of using such formulations.

In one general aspect, the application relates to a pharmaceuticalformulation, the formulation comprising:

-   -   a) a fusion protein in a concentration of about 0.5 mg/mL to        about 120 mg/mL,    -   b) a polyol or alcohol selected from a group consisting of        sucrose, trehalose, mannitol, sorbitol, benzyl alcohol,        polyvinyl alcohol, polyethylene glycol (PEG) 400-12000, in a        concentration of about 1% to about 10% w/v,    -   c) a buffering agent selected from a group consisting of sodium        phosphate, histidine, sodium citrate, sodium acetate, sodium        bicarbonate, and trisodium citrate dihydrate in a concentration        of about 10 mM to about 50 mM, and    -   d) a surfactant in a concentration of about 0.01 to about 4%        w/v,        wherein the formulation is at a pH of about 5.5-7.5 and        optionally, the formulation further comprises a polysaccharide        selected from the group consisting of sodium        carboxymethylcellulose, microcrystalline cellulose, or sodium        hyaluronate.

According to embodiments of the application, the surfactant is selectedfrom a group consisting of polysorbate 20, polysorbate 80 and poloxamer188, preferably polysorbate 20.

According to embodiments of the application, the surfactant is in aconcentration of about 0.03%.

According to embodiments of the application, the fusion protein is in aconcentration of about 1 mg/mL to about 90 mg/mL, preferably about 20mg/mL to about 80 mg/mL, more preferably the fusion protein is in aconcentration of about 40 mg/mL.

According to embodiments of the application, the polyol is trehalose ina concentration of about 25 mM to about 250 mM, preferably about 190 mM.

According to embodiments of the application, the buffering agent ishistidine in a concentration of about 10 mM to about 40 mM, preferablyabout 20 mM to about 30 mM, more preferably the histidine is in aconcentration of about 25 mM.

According to embodiments of the application, the fusion proteincomprises, from N-terminus to C-terminus in the following order:

-   -   a) an extracellular domain of a Vascular Endothelial Growth        Factor receptor (VEGFR);    -   b) an Fc domain of human immunoglobulin G; and    -   c) an integrin binding protein or its fragment thereof.

According to embodiments of the application, the fusion proteincomprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, or SEQ ID NO: 18.

According to embodiments of the application, the pH is about 5.5 toabout 7.0, preferably the pH is about 6.0.

According to embodiments of the application, the formulation is stableat −70° C., −20° C. and/or 5° C. for at least 24 months.

According to embodiments of the application, the formulation retainsprotein purity and potency after least 9 months at −70° C., −20° C.and/or 2-8° C., preferably at 2-8° C.

According to embodiments of the application, the formulation furthercomprises a salt in a concentration of about 10 mM to 50 mM.

According to embodiments of the application, the formulation furthercomprises at least one amino acid in a concentration of about 10 mM to50 mM.

According to embodiments of the application, the salt is selected fromsodium chloride, magnesium chloride, calcium chloride, or potassiumchloride.

According to embodiments of the application, the amino acid is selectedfrom the group consisting of arginine, methionine, proline, histidine,cysteine, lysine, glycine, aspartate, tryptophan, glutamate, andisoleucine.

According to embodiments of the application, the pharmaceuticalformulation can be used in a method of treating an ocular disease.

According to embodiments of the application, the ocular disease isselected from neovascularization or ischemia uveitis, retinalvasculitis, angioid streaks, retinitis pigmentosa, cornealneovascularization, iris neovascularization, neovascularizationglaucoma, post-surgical fibrosis in glaucoma, proliferativevitreoretinopathy (PVR), choroidal neovascularization (CNV), optic discneovascularization, retinal neovascularization, vitrealneovascularization, pannus, pterygium, vascular retinopathy, diabeticretinopathy (DR, non-proliferative and proliferative DR) without DME,diabetic retinopathy (DR, non-proliferative and proliferative DR) withDME, diabetic macular edema (DME), exudative (wet) and non-exudative(dry) age-related macular degeneration (AMD), macular edema, macularedema following retinal vein occlusion (RVO), retinal vein occlusion(RVO), central retinal vein occlusion (CRVO), Branch retinal veinocclusion (BRVO), Retinal Angiomatous Proliferation (RAP), polypoidalchoroidal vascularization (PCV), vitreomacular adhesion (VMA) and/orvitreomacular traction (VMT).

According to embodiments of the application, the formulation isadministered at a dose of about 0.03-10 mg per eye, preferably about3.0-6.0 mg per eye, more preferably the formulation is administered at adose of about 4 mg per eye.

According to embodiments of the application, the formulation isadministered at a dose of about 4 mg per eye.

Another general aspect of the application relates to a pharmaceuticalformulation, the formulation comprising:

-   -   a) a fusion protein in a concentration of 40 mg/mL,    -   b) 25 mM histidine,    -   c) 190 mM trehalose, sucrose, or mannitol,    -   d) 0.03% polysorbate 20 or polysorbate 80,        wherein the formulation is at a pH of about 6.0.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects, features, and advantages ofexemplary embodiments will become more apparent and may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings.

FIG. 1 is a graph of the mean percent change from baseline of thevascular leakage area following a single intravitreal injection ofFusion Protein 1 at 0.6, 1.0, and 1.9 mg/eye in a laser-inducedchoroidal neovascularization model in rhesus monkeys. Data presented asmean±SEM. All doses were administered as a single intravitreal dose onDay 0. Lucentis® (0.5 mg) was used as system suitability and positivecontrol. All spots analyzed were of Grade III/IV. % VascularLeakage=(Baseline leakage area−Treatment leakage area)÷(Baseline leakagearea)×100%; Statistical analysis was conducted to compare treatmentgroup with vehicle group by Mann-Whitney U test, *, p<0.05. ^(a)In the1.9 mg group, data from one monkey with severe ocular inflammation wasremoved which affected statistical significance (p=0.057). n=4 eyes pergroup.

FIG. 2 is a graph of the mean retinal thickness assess by opticalcoherence tomography following a single intravitreal injection of Fusionprotein 1 at 0.6, 1.0, and 1.9 mg/eye in a laser-induced choroidalneovascularization model in rhesus monkeys. Data presented as mean±SEM.All doses were administered as a single intravitreal dose on Day 0.Lucentis® (0.5 mg) was used as system suitability and positive control.All spots analyzed were of Grade III/IV. % Retinal thickness=(Baselineretinal thickness−Treatment retinal thickness)÷(Baseline retinalthickness−Pre-study retinal thickness)×100%; Statistical analysis wasconducted to compare treatment group with vehicle group by Mann-WhitneyU test, *, p<0.05. ^(a)In the 1.9 mg group, data from one monkey withsevere ocular inflammation was removed which affected statisticalsignificance (p=0.057). n=4 eyes per group.

FIG. 3 is a graph of Masson's Trichrome staining of grade III/IV lesionsof a laser-induced choroidal neovascularization model in rhesus monkeystreated as indicated. Data presented as mean±SEM. Enucleation wasperformed on Day 29. Selected Grade 4 lesion spots were analyzed byMasson's Trichrome staining. Student's t-test analysis was conducted tocompare treatment group with vehicle group, *, p<0.05. n=4 eyes pergroup.

FIG. 4 is a graph of lung hydroxyproline levels in a belomycine-inducedC57BL/6 mouse lung fibrosis model following treatment as indicated (n=8animal for each treatment group and n=4 for the sham control group).$p<0.05, treated vs. sham control, unpaired Student's t-test; *p<0.05,treated vs. vehicle, One-way ANOVA and Dunnett's test. In the FusionProtein 1 group, one animal data was not available due to early deathwith unknown reason on Day 3.

FIGS. 5A-5F are plots of the diffusion coefficients of Fusion Protein 1in (FIG. 5A) phosphate buffer pH 7.0; (FIG. 5B) phosphate buffer pH 6.5;(FIG. 5C) histidine buffer pH 6.5; (FIG. 5D) histidine buffer pH 6.0;(FIG. 5E) citrate buffer pH 6.0; and (FIG. 5F) citrate buffer pH 5.5,respectively. The diffusion coefficients were determined by dynamiclight scattering (DLS).

FIGS. 6A-6B are graphs of the turbidity measurement analyzed at twowavelengths for both 40 and 80 mg/L of Fusion Protein 1. FIG. 6A depictswavelength 660 nm and FIG. 6B depicts wavelength 320 nm. NaPi=Sodiumphosphate, Cit=Citrate and His=Histidine.

FIGS. 7A-7E are non-reducing SDS-PAGE with Coomassie Blue stained gelsof Fusion Protein 1. FIGS. 7A and 7B include Fusion Protein 1 at 40 or80 mg/mL incubated in citrate buffer at 4° C. or 40° C. for a period,beginning (lanes 2 and 3), 4 days (lanes 4 and 5), 7 days (lanes 6 and7), and 14 days (lanes 8 and 9). FIGS. 7C and 7D include Fusion Protein1 at 40 or 80 mg/mL incubated in histidine buffer at 4° C. or 40° C. fora period, beginning (lanes 2 and 3), 4 days (lanes 4 and 5), 7 days(lanes 6 and 7), and 14 days (lanes 8 and 9). FIG. 7E includes FusionProtein 1 at 40 or 80 mg/mL incubated in histidine or citrate buffer at4° C. or 40° C. on Day 28. NaPi=Sodium phosphate, Cit=Citrate andHis=Histidine. For each sample well, 3 μg of protein was loaded.

FIGS. 8A-8E are reducing SDS-PAGE gels with Coomassie Blue stained ofFusion Protein 1. FIGS. 8A and 8B include Fusion Protein 1 at 40 or 80mg/mL incubated in citrate buffer at 4° C. or 40° C. for a period,beginning (lanes 2 and 3), 4 days (lanes 4 and 5), 7 days (lanes 6 and7), and 14 days (lanes 8 and 9). FIGS. 8C and 8D include Fusion Protein1 at 40 or 80 mg/mL incubated in histidine buffer at 4° C. or 40° C. fora period, beginning (lanes 2 and 3), 4 days (lanes 4 and 5), 7 days(lanes 6 and 7), and 14 days (lanes 8 and 9). FIG. 8E includes FusionProtein 1 at 40 or 80 mg/mL incubated in histidine or citrate buffer at4° C. or 40° C. on Day 28. NaPi=Sodium phosphate, Cit=Citrate andHis=Histidine. For each sample well, 3 μg of protein was loaded.

FIG. 9 is a graph of the thermal stability of Fusion Protein 1 at 1mg/mL formulated in 25 mM histidine buffer, 190 mM trehalose, 0.03% PS20at pH 6.0 and determined by Differential Scanning calorimeter (DSC).

FIG. 10 is a graph of a single dose administration of Fusion Protein 1at the indicated concentrations in Dutch Belted rabbits, which inhibitshuman VEGF-A₁₆₅ induced retinal vascular permeability.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by a person skilled in theart to which this invention belongs.

As used herein, the singular forms “a”, “an”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a binding domain” includes a plurality of bindingdomains and equivalents thereof known to those skilled in the art.

As used herein, the term “polypeptide” and “protein” may be usedinterchangeably to refer to a long chain of peptide having an amino acidsequence of the native protein or the amino acid sequence with one ormore mutations such as deletions, additions, and/or substitutions of oneor more amino acid residues.

A “fusion protein” refers to a protein having two or more portionscovalently linked together, where each of the portions is derived fromdifferent proteins.

The present invention provides a pharmaceutical formulation comprising afusion protein comprising an integrin binding peptide selected from agroup consisting of disintegrin (see U.S. Pat. No. 7,943,728 and PCTApplication No. PCT/US15/46322 for the description of amino acidsequences, each of which is incorporated by reference in its entirety),anti-integrin αvβx antibody (see U.S. Pat. Nos. 6,160,099 and 8,350,010for the description of amino acid sequences, each of which isincorporated by reference in its entirety), anti-integrin α5β1 antibody,fibronectin (see U.S. Pub. No. 2015/0218251 for the description of aminoacid sequences, which is incorporated by reference in its entirety)targeting integrin isoform αvβx or α5β1 and their integrin bindingfragments, other protein binding peptide targeting an angiogenic factorand a Fc domain, wherein x is 1, 3, 5, 6 or 8.

The term “antibody”, as used herein, is intended to refer toimmunoglobulin molecules comprised of four polypeptide chains, two heavychains and two light chains inter-connected by disulfide bonds. Afull-length heavy chain includes a variable region domain, VH, and threeconstant region domains, CH1, CH2 and CH3. The VH domain is at theamino-terminus of the polypeptide, and the CH3 domain is at thecarboxy-terminus. A full-length light chain includes a variable regiondomain, VL, and a constant region domain, CL. An antigen bindingfragment (Fab) is comprised of one light chain and the CH1 and variableregions of one heavy chain. The heavy chain of a Fab molecule cannotform a disulfide bond with another heavy chain molecule. A Fab′ fragmentcontains one light chain and one heavy chain that contains more of theconstant region, between the CH1 and CH2 domains, such that aninterchain disulfide bond can be formed between two heavy chains to formdiabodies. A variable fragment (Fv) region comprises the variableregions from both the heavy and light chains but lacks the constantregions. Single-chain fragments (scFv) are Fv molecules in which theheavy and light chain variable regions have been connected by a flexiblelinker to form a single polypeptide chain which forms an antigen-bindingregion. Single chain antibodies are discussed in detail in WO88/01649and U.S. Pat. Nos. 4,946,778 and 5,260,203. As used herein, the term“antibody” includes an immunoglobulin molecule with two full lengthL-chains and two full length H-chains, and fragments thereof, such as anantigen binding fragment (Fab), a Fv region, a scFv, etc.

The term “Fc domain” refers to a molecule or sequence comprising thesequence of a non-antigen binding portion of an antibody, whether inmonomeric or multimeric form. The original immunoglobulin source of anFc is preferably of human origin and can be from any isotype, e.g., IgG,IgA, IgM, IgE or IgD. A full-length Fc consists of the following Igheavy chain regions: the flexible hinge region between CH1 and CH2, CH2and CH3, wherein the two chains are typically connected by disulfidebonds in the flexible hinge region.

The present invention provides a fusion protein comprising an integrinbinding peptide that includes disintegrin and its integrin bindingfragments, other protein binding peptide comprising an extracellulardomain of VEGF receptor and a Fc domain, wherein the integrin bindingpeptide comprises at least one mutation on or adjacent to the RGD motif.In accordance with embodiments of the present invention, the disintegrinand its integrin binding fragments have an amino acid sequence selectedfrom a group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, or amino acidsequence having at least 85% sequence identity to SEQ ID NO: 1, SEQ IDNO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQID NO: 7.

As used herein, “disintegrin” refers to a class of cysteine-richproteins or polypeptides that are potent soluble ligands of integrins.The RGD motif is a tri-peptide (Arg-Gly-Asp) conserved in most monomericdisintegrins and is located at an integrin-binding loop. Thedisintegrins described herein are isolated from snake venom or derivedfrom wild-type forms and have at least one mutation on or adjacent to aRGD motif to selectively bind to or target to various integrin isoforms.The term “adjacent to a RGD motif” as used herein means any mutationwhich occurs at any amino acid residue within 15-20 amino acids from theRGD motif in a given peptide, polypeptide, protein sequence.

Other amino acid sequence variants of the disintegrin are alsocontemplated. For example, binding affinity and/or other biologicalproperties of a disintegrin can be improved by altering the amino acidsequence encoding the protein. Disintegrin mutants can be prepared byintroducing appropriate modifications into the nucleic acid sequenceencoding the protein or by introducing modification by peptidesynthesis. Such modifications include mutations such as deletions from,insertions into, and/or substitutions within the nucleic or amino acidsequence of the disintegrin. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final amino acid construct ofthe disintegrin provided that the final construct possesses the desiredcharacteristics such as binding to an integrin superfamily member and/orinhibiting the integrin activated pathway.

Substantial modifications in the biological properties of the proteinsor polypeptides are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain.

A useful method for identifying certain residues or regions of thefusion protein that are preferred locations for mutagenesis is known as“alanine scanning mutagenesis” as described in Cunningham B C et al.Science. 1989; 244:1081-1085. For example, a residue or group of targetresidues are identified (e.g., charged residues such as Arg, Asp, His,Lys and Glu) and replaced by a neutral (most preferably glycine, alanineor leucine) or oppositely charged amino acid (from positive charge tonegative charge or vice versa) to affect the interaction of the aminoacids with the target binding partner. Those amino acid locationsdemonstrating functional sensitivity to the substitutions then arerefined by introducing further or other variants at, or for, the sitesof substitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, random mutagenesis may be conducted at thetarget codon or region and the expressed fusion polypeptide variants arescreened for desired activity. For example, cysteine bond(s) may beadded to the fusion protein or protein components to improve itsstability.

Accordingly, provided herein are disintegrin mutants that can be acomponent of any fusion protein disclosed herein. In some embodiments,the disintegrin comprises an amino acid sequence with at least 85%, atleast 90%, at least 95%, or at least 99% sequence identity to the aminoacid sequence of disintegrins selected from a group consisting ofRhodostomin (SEQ ID NO: 1), Triflavin (SEQ ID NO: 3), Echistatin (SEQ IDNO: 4), Trimucrin (SEQ ID NO: 5), Elegantin (SEQ ID NO: 6) and Trigramin(SEQ ID NO: 7). In some embodiments, a disintegrin comprises an aminoacid sequence having at least one mutation on or adjacent to the RGDmotif of Rhodostomin (SEQ ID NO: 1), Triflavin (SEQ ID NO: 3),Echistatin (SEQ ID NO: 4), Trimucrin (SEQ ID NO: 5), Elegantin (SEQ IDNO: 6) or Trigramin (SEQ ID NO: 7). In some embodiments, the disintegrincomprises an amino acid sequence with at least 85%, at least 90%, atleast 95%, or at least 99% sequence identity to the amino acid sequenceof a disintegrin mutant (SEQ ID NO: 2). The Xaa in SEQ ID NO: 2indicates various positions that can be modified by either insertion,substitution or deletion to produce amino acid sequence variants thatare different from the wild type form of disintegrin. According to someexamples, the Xaa at position 50 of SEQ ID NO: 2 which corresponds toglycine (Gly) in the RGD motif of wild type Rhodostomin (SEQ ID NO: 1)may be substituted with naturally occurring amino acids other thanglycine to generate Rhodostomin mutants. In other examples, one or moreXaa in SEQ ID NO: 2 may also be substituted with naturally occurringamino acids other than those originally found in corresponding positionsof wild type Rhodostomin (SEQ ID NO: 1) to generate various Rhodostominmutants. It is further noted that the disintegrin mutants are notlimited to include only single mutation at any Xaa in SEQ ID NO: 2,multiple mutations that occur at several locations of Xaa in SEQ ID NO:2 or corresponding locations in other consensus sequences of disintegrin(such as SEQ ID NOs: 3-7) may also be encompassed by the scope of theinvention.

Rhodostomin mutants have been described in U.S. Pat. No. 7,943,728 andPCT Application No. PCT/US15/46322 and their sequences are incorporatedherein by reference. For example, PCT/US15/46322 describes thedisintegrin variant comprised of a mutant RGD loop having the amino acidsequence selected from the group consisting of SEQ ID NO: 24 to SEQ IDNO: 26, and at least one of a mutant linker having the amino acidsequence selected from the group consisting of SEQ ID NO: 29 to SEQ IDNO: 41, and a mutant C-terminus having the amino acid sequence selectedfrom the group consisting of SEQ ID NO: 42 to SEQ ID NO: 47. Morepreferably, the disintegrin variant comprises the mutant RGD loop, themutant linker and the mutant C-terminus described herein.

Mutants of Rhodostomin or disintegrins with one or more modifications inaddition to the RGD motif, e.g., in the linker region or the C-terminus,exhibited the capability to selectively bind to αvβ3, αvβ5, αvβ6, α5β1or αIIbβ3. For example, Rhodostomin variants with the mutation in thelinker region (39X40X41X42X43X), in which the SRAGK (SEQ ID NO: 50) wasreplaced by KKKRT (SEO ID NO: 51), KKART (SEO ID NO: 52), MKKGT (SEO IDNO: 53) IEEGT (SEO ID NO: 54), LKEGT (SEO ID NO: 55), AKKRT (SEO ID NO:56), KAKRT (SEO ID NO: 57), KKART (SEO ID NO: 58), KKKAT (SEO ID NO:59), KKKRA (SEO ID NO: 60), KAKRA (SEO ID NO: 61), or SKAGT (SEO ID NO:62) amino acids, had their highest effects on integrins in the followingorder: αIIbβ3 (^(˜)2-fold)>α5β1 (^(˜)5-fold)>αvβ3 (^(˜)14-fold).

Rhodostomin variants with the mutation in the C-terminal region(66X67X68X69X70X), in which the RYH was replaced by RYH (SEO ID NO: 63),RNGL (SEQ ID NO: 64), RGLYG (SEO ID NO: 65), RGLY (SEO ID NO: 66), RDLYG(SEO ID NO: 67), RDLY (SEO ID NO: 68), RNGLYG (SEO ID NO: 69), or RNPWNG(SEO ID NO: 70) amino acids, had their highest effects on integrins inthe following order: αIIbβ3 (^(˜)13-fold)>αvβ5 (^(˜)8-fold)=αvβ6(^(˜)8-fold)>αvβ3 (^(˜)4-fold)>α5β1 (^(˜)2-fold). Table 1 shows thesequences of SEQ ID NOs: 24 to 49 and their corresponding positions onSEQ ID NO: 1.

TABLE 1 SEQ Position on ID NO Sequence SEQ ID NO: 1 24 RIARGDNP 46-53 25RRARGDNP 26 ARGRGDNP 27 ARGRGDDL 28 ARARGDNP 29 KKKRTIC 39-45 30 MKKGTIC31 IEEGTIC 32 KGAGKIC 33 LKEGTIC 34 AKKRTIC 35 KAKRTIC 36 KKARTIC 37KKKATIC 38 KKKRAIC 39 KAKRAIC 40 SKAGTIC 41 KKKRTIC 42 PRWNDL 65-68 43PRNGLYG 44 PGLYG 45 PDLYG 46 PPLYG 47 PRLYG 48 PELYG 49 PYLYG

Although the variants of disintegrins are discussed mostly withreference to the amino acid sequences discussed above, polypeptidesequences or nucleotide sequence encoding the snake venom such asAlbolabrin, Applagin, Basilicin, Batroxostatin, Bitistatin, Cereberin,Cerastin, Crotatroxin, Durissin, Flavoridin, Flavostatin, Halysin,Halystatin, Jararacin, Jarastatin, Kistrin, Lachesin, Lutosin, Molossin,Salmosin, Saxatilin, Tergeminin, Trimestatin, Trimutase, Ussuristatin,Viridian and their mutants having at least one mutation on or adjacentto the RGD motif may also be encompassed by the scope of the presentinvention.

Without being bound by theory, it is contemplated herein thatdisintegrins inhibit the integrin activated pathway by binding to anintegrin superfamily member to block its interaction with a multivalentintegrin receptor. In some aspects, the disintegrin binds to an integrinsuperfamily member which includes but is not limited to the integrinisoforms αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, α5β1 and/or αIIbβ3.

According to the present invention, the other protein binding peptide ofthe fusion protein may be receptor protein that binds to a targetselected from the group consisting of a tumor antigen, a TNF receptorsuperfamily member, a Hedgehog family member, a receptor tyrosinekinase, a proteoglycan-related molecule, a TGF-beta superfamily member,a Wnt-related molecule and an angiogenesis target.

According to some embodiments of the invention, the other proteinbinding peptide may specifically bind to an angiogenesis target whichincludes but is not limited to Angiopoietin (ANG), Ephrin (Eph),Fibroblast Growth Factor (FGF), Neuropilin (NRP), PlasminogenActivators, Platelet-Derived Growth Factor (PDGF), Tumor Growth factorbeta (TGF-β), Vascular Endothelial Growth Factor (VEGF), VascularEndothelial cadherin (VE-cadherin), Tumor necrosis factor-alpha (TNF-α),Insulin like growth factor (IGF-1) and their receptors. Therefore, inaccordance with embodiments of the invention, the other protein bindingpeptide may include extracellular portions of a receptor protein thatbinds to and antagonizes the angiogenesis target. In other embodiments,the other protein binding peptide may bind to extracellular portions ofangiogenic factor receptors.

In some embodiments, the other protein binding peptide may be ananti-VEGF antibody (see WO2015/200905 for the description of its aminoacid sequence, which is incorporated herein by reference in itsentirety) that binds to the VEGF ligand or an anti-VEGFR1 or anti-VEGFR2antibody (see U.S. Pat. No. 5,874,542 for the description of its aminoacid sequence, which is incorporated herein by reference in itsentirety) that binds to VEGF receptor. In other embodiments, the otherprotein binding peptide may also be an anti-PDGF antibody (see U.S. Pat.No. 5,094,941 for the description of its amino acid sequence, which isincorporated herein by reference in its entirety) that binds to the PDGFligand or an anti-PDGFRβ antibody (see U.S. Pat. No. 9,265,827 for thedescription of its amino acid sequence, which is incorporated herein byreference in its entirety for all purposes) that binds to PDGF receptor.

In certain embodiments, the other protein binding peptide binds to thesame VEGF as any one of VEGF receptors (VEGFR): VEGFR1, VEGFR2 andVEGFR3. In some embodiments, the other protein binding peptide comprisesat least one extracellular portion of a VEGFR of any of the VEGFRsdescribed herein. For example, the other protein binding peptidecomprises at least one extracellular portion of VEGFR1 or oneextracellular portion of VEGFR2. In another example, the other proteinbinding peptide comprises one extracellular portion of VEGFR1 such asIg-like domain 2 (D2) and one extracellular portion of VEGFR2 such asIg-like domain 3 (D3). In some aspect, the other protein binding peptidecomprises one extracellular portion of a VEGFR1 comprising amino acidsequence of SEQ ID NO: 8 and one extracellular portion of a VEGFR2comprising amino acid sequence of SEQ ID NO: 9. In some aspect, theother protein binding peptide comprises a fusion of extracellularportions of VEGFR1 and VEGFR2 comprising an amino acid sequence of SEQID NO: 10 or an amino acid sequence with at least 85% sequence identityto SEQ ID NO: 10.

In other embodiments, the other protein binding peptide binds to thesame PDGF as any one of PDGF receptors (PDGFR): PDGFRα and PDGFRβ. Insome embodiments, the other protein binding peptide comprises at leastone extracellular portion of a PDGFR of any of the PDGFRs describedherein. For example, the other protein binding peptide comprises atleast one extracellular portion of PDGFRα or one extracellular portionof PDGFRβ. In another example, the other protein binding peptidecomprises one extracellular portion of PDGFRβ such as Ig-like domain1-3. In some aspect, the other protein binding peptide comprises anextracellular portion of a PDGFR comprising an amino acid sequence ofSEQ ID NO: 11 or an amino acid sequence with at least 85% sequenceidentity to SEQ ID NO: 11.

In accordance with other embodiments, the invention also provides afusion protein comprising an integrin binding peptide that includes anamino acid sequence of SEQ ID NO:1 with at least one mutation on oradjacent to the RGD motif, an amino acid sequence having at least 85%sequence identity to SEQ ID NO: 1 or an amino acid sequence of SEQ IDNO: 2, a human or humanized constant sub-region comprising animmunoglobulin CH2 domain and a CH3 domain, and other protein bindingpeptide having an Ig-like D2 of a VEGFR1 and an Ig-like D3 of a VEGFR2.In a further embodiment of the fusion protein, the integrin bindingpeptide has at least 85% sequence identity to SEQ ID NO: 2.

The term “percent (%) sequence identity” with respect to a referencepolypeptide or nucleic acid sequence is defined as the percentage ofamino acid residues or nucleotides in a candidate sequence that areidentical with the amino acid residues or nucleotides in the referencepolypeptide or nucleic acid sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid or nucleic acid sequence identity can be achieved in variousways that are within the skill in the art, for instance, using publiclyavailable computer software programs, for example, as those described inCurrent Protocols in Molecular Biology (Ausubel et al. eds., 1987), andincluding BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) Software. Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared. For purposesherein, the % amino acid sequence identity of a given amino acidsequence A to, with or against a given amino acid sequence B iscalculated as follows: 100 times the fraction X/Y, where X is the numberof amino acid residues score as identical matches by the sequencealignment program in that program's alignment of A and B, and where Y isthe total number of amino acid residues in B. It will be appreciatedthat where the length of amino acid sequence A is not equal to thelength of amino acid sequence B, the % amino acid sequence identity of Ato B will not equal the % amino acid sequence identity of B to A.

The present invention provides a dimeric fusion protein comprising twofusion proteins, wherein each fusion protein comprises any fusionprotein disclosed herein. In one embodiment, the dimeric fusion proteincomprises two identical fusion proteins. In another embodiment, thedimeric fusion protein may comprise two different fusion proteins. Thefusion proteins disclosed herein may form multimers of two or moreidentical fusion proteins or form heterologous fusion proteins through amultimerization domain which includes a constant sub-region of a humanor humanized antibody. In some embodiments, the constant sub-region of ahuman or humanized antibody is selected from the group consisting of anIgG Fc region, IgA Fc region, IgM Fc region, IgD Fc region and IgE Fcregion. In the further embodiment, the constant sub-region of a human orhumanized antibody is selected from the group consisting of an IgG1 Fcregion, IgG2 Fc region, IgG3 Fc region and IgG4 Fc region. In someaspect, the sub-region comprises a CH2 region and a CH3 region of IgG1,IgG2, IgG3, or IgG4. Amino acid sequences encoding immunoglobulins thatcomprise Fc regions are well known in the art.

The components of the fusion protein may be connected directly to eachother or be connected via linkers. Generally, the term “linker” meansone or more molecules e.g., nucleic acids, amino acids or non-peptidemoieties which may be inserted between one or more component domains.For example, linkers may be used to provide a desirable site of interestbetween components for ease of manipulation. A linker may also beprovided to enhance expression of the fusion protein from a host cell,to decrease steric hindrance such that the component may assume itsoptimal tertiary structure and/or interact appropriately with its targetmolecule. A linker sequence may include one or more amino acidsnaturally connected to a receptor component or may be an added sequenceused to enhance expression of the fusion protein, to providespecifically desired sites of interest, to allow component domains toform optimal tertiary structures and/or to enhance the interaction of acomponent with its target molecule.

Preferably, the linker increases flexibility of the fusion proteincomponents without interfering with the structure of each functionalcomponent within the fusion protein. In some embodiments, the linkermoiety is a peptide linker with a length of 2 to 100 amino acids.Exemplary linkers include linear peptides having at least two amino acidresidues such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala, Gly (G)n and Gly-Ser(GS) linker. The GS linker described herein includes but is not limitedto (GS)n, (GSGSG)n, (G₂S)n, G₂S₂G, (G₂SG)n, (G₃S)n, (G₄S)n, (GGSGG)nGnand GSG₄SG₄SG, wherein n is 1 or more. One example of the (G)n linkerincludes a G₉ linker. Suitable linear peptides include polyglycine,polyserine, polyproline, polyalanine and oligopeptides consisting ofalanyl and/or serinyl and/or prolinyl and/or glycyl amino acid residues.The linker moieties may be used to link any of the components of thefusion proteins disclosed herein. In some embodiments, a linker is usedbetween an extracellular portion of a receptor protein and a constantsub-region of an immunoglobulin. In other embodiments, a linker is usedbetween disintegrin or its variant and a constant sub-region of animmunoglobulin. In certain embodiments, the fusion protein comprises alinker between an extracellular portion of a receptor protein anddisintegrin or its variant, and a linker between disintegrin or itsvariant and a constant sub-region of an immunoglobulin. As embodied inthe present invention, a fusion protein may comprise at least one linkerbut no more than four linkers.

The fusion protein described herein may or may not comprise a signalpeptide that functions for secreting the fusion protein from a hostcell. A nucleic acid sequence encoding the signal peptide can beoperably linked to a nucleic acid sequence encoding the protein ofinterest. In some embodiments, the fusion protein comprises a signalpeptide. In some embodiment, the fusion protein does not comprise asignal peptide.

Moreover, the fusion proteins described in the present invention maycomprise modified forms of the protein binding peptides. For example,the fusion protein components may have post-translational modifications,including for example, glycosylation, sialylation, acetylation, andphosphorylation to any of the protein binding peptides.

Although the embodiments are generally described with reference to twoprotein binding peptides included in the fusion protein, the inventionalso contemplates a fusion protein which incorporates more than twoprotein binding peptides to provide any additional or synergisticeffects in terms of inhibiting the process of angiogenesis. For example,there may be an additional protein binding peptide that binds to otherangiogenesis targets or acts as angiogenic factor antagonists to belinked to the existing two protein binding peptides.

Fusion proteins for the pharmaceutical formulations disclosed herein maybe purified and identified using commonly known methods such asfractionation on immunoaffinity or ion-exchange columns; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on acation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, Sephadex G-75;hydrophobic affinity resins, ligand affinity using a suitable bindingpartner immobilized on a matrix, centrifugation, Enzyme-LinkedImmunosorbent Assay (ELISA), BIACore, Western Blot assay, amino acid andnucleic acid sequencing, and biological activity. In some embodiments,the fusion protein is expressed in host cells and purified therefromusing a combination of one or more standard purification techniques,including, but not limited to Protein A affinity chromatography, ProteinG affinity chromatography, buffer exchange, size exclusionchromatography, ultrafiltration, and dialysis. Accordingly, therecovered fusion protein is substantially pure. In a further embodiment,the recovered fusion protein is at least any of 90%, 95%, 96%, 97%, 98%or 99% pure.

The fusion proteins or fusion protein components disclosed herein may becharacterized or assessed for biological activities including, but notlimited to, affinity to a target binding partner, competitive binding,inhibitory activity, inhibition of cell proliferation, inhibition oftumor growth, and inhibition of angiogenesis. In some embodiments, thefusion proteins or fusion protein components disclosed herein can beassessed for biological activity in vitro and in vivo. Many methods forassessing binding affinity are known in the art and can be used toidentify the binding affinities of fusion proteins or fusion proteincomponents to a binding partner through a titration method. Bindingkinetics can be expressed as the steady-state equilibrium bindingconstant, expressed as the dissociation constant (K_(D)) or half maximaleffective concentration (EC₅₀) values.

In certain embodiments, a fusion protein has an EC₅₀ of less than orequal to 1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, or 0.001 nM forinhibition of an activity (e.g., inhibition of angiogenic factoractivity and/or integrin activity). In any of the embodiments herein, afusion protein has a K_(D) for a binding partner (angiogenic factorand/or integrin) of less than about 1.0 mM, 500 μM, 100 μM, 50 μM, 10μM, 5 μM, 1 μM, 500 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 100pM, 50 pM, 10 pM or 5 pM, including any values in between these numbers.

In certain embodiments, the isoelectric point (pI) of the fusion proteinis about 4.0 to 9.0. In certain embodiments, the isoelectric point (pI)of the fusion protein is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3,7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 9.3, 8.4, 8.5, 8.6, 8.7,8.8, 8.9, 9.0, or any value in between. Preferably, the isoelectricpoint (pI) of the fusion protein is 8.17.

The invention also provides a pharmaceutical composition comprising afusion protein comprising an integrin binding peptide selected from agroup consisting of disintegrin, anti-integrin αvβx antibody,anti-integrin α5β1 antibody, fibronectin targeting integrin αvβx or α5β1and their integrin binding fragments, other protein binding peptidetargeting an angiogenic factor and a Fc domain, wherein x is 1, 3, 5, 6or 8. Compositions of the invention comprise a therapeutically effectiveamount of the fusion protein.

The term “therapeutically effective amount” means an amount of atherapeutically active compound needed to elicit the desired biologicalor clinical effect. According to embodiments of the invention, “atherapeutically effective amount” is an amount sufficient to effectbeneficial or desired results, including clinical results. Atherapeutically effective amount can be administered in one or moreadministrations. In terms of a disease state, a therapeuticallyeffective amount is an amount sufficient to ameliorate, stabilize, ordelay development of a disease. According to specific embodiments of theinvention, a therapeutically effective amount is an amount of a fusionprotein needed to treat or prevent a disorder characterized by abnormalangiogenesis, such as a disease characterized by neovascularization,vascular permeability, edema, inflammation, retinopathies, fibrosis orcancer.

The term “potency” means the ability of the fusion protein to functionas intended at the clinical dose administered.

The term “pharmaceutical formulation” herein refers to formulationsincluding pharmaceutically acceptable carriers, e.g., used to administerVEGF receptor fusion proteins (e.g., aflibercept or conbercept) to asubject for a therapeutic/medicinal use.

The term “purity” herein refers to the absence of contamination of thefusion protein. Contaminants as referred to herein include proteinspecies other than the intended fusion protein molecule, arising fromthe compound manufacturing process as impurities and/or degradation ofthe manufactured protein compound.

In some embodiments, the pharmaceutical composition comprising a fusionprotein comprises a fusion protein formulated in a buffer at a proteinconcentration from about 0.5 to about 100 mg/mL, preferably about 40 toabout 80 mg/mL, such as about 40, 50, 60, 70 or 80 mg/mL, mostpreferably about 40±about 4 mg/mL. In other preferred embodiments, thefusion protein is formulated in a buffer at a protein concentration ofmore than about 40 mg/mL.

In particular embodiments, the buffer is a buffer with a pH of about 5.5to 7.0, more preferably about 6.0 to 6.5, even more preferably about6.0. In particular embodiments, the buffer is a phosphate buffer with apH of about 6.5 to 8, more preferably about 7 to 7.5, even morepreferably about 7.2. The phosphate buffer comprises about 5 to 20 mMsodium phosphate, such as 5, 10, 15 or 20 mM sodium phosphate, morepreferably about 10 mM sodium phosphate; about 20 to 60 mM sodiumchloride, more preferably about 40 mM sodium chloride; about 1 to 10%weight-per-volume (w/v) sucrose, more preferably about 5% w/v sucrose;and about 0.01 to 0.05% w/v of a surfactant, more preferably about 0.03%w/v polysorbate 20.

In particular embodiments, the pharmaceutical formulation comprises apolyol or alcohol selected from a group consisting of sucrose,trehalose, mannitol, sorbitol, benzyl alcohol, polyvinyl alcohol,polyethylene glycol (PEG) 400-12000, in a concentration of about 1% toabout 10% w/v such as about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%w/v.

In preferred embodiments, the polyol is trehalose in a concentration ofabout 25 mM to about 250 mM, such as about 25 mM, 50 mM, 75 mM, 100 mM,125 mM, 150 mM, 175 mM, 200 mM, 225 mM, or 250 mM. In other preferredembodiments, the polyol is trehalose in a concentration of about 100 to200 mM such as about 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160mM, 170 mM, 180 mM, 190 mM, or 200 mM. In other preferred embodiments,the polyol is trehalose in a concentration of about 190 mM.

In particular embodiments, the pharmaceutical formulation comprises abuffering agent. In particular embodiments, the buffering agent isselected from a group consisting of sodium phosphate, histidine, sodiumcitrate, sodium acetate, sodium bicarbonate, and trisodium citratedihydrate in a concentration of about 10 mM to about 50 mM such as about10 mM, 20 mM, 30 mM, 40 mM, or 50 mM. In preferred embodiments, thebuffering agent is in a concentration of about 10 mM to about 40 mM suchas about 10 mM, 20 mM, 30 mM, or 40 mM. In other preferred embodiments,the buffering agent is in a concentration of about 20 mM to 30 mM suchas about 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM,29 mM, or 30 mM. In other preferred embodiments, the buffering agent isin a concertation of about 25 mM.

In particular embodiments, the pharmaceutical formulation furthercomprises a polysaccharide selected from the group consisting of sodiumcarboxymethylcellulose, microcrystalline cellulose, or sodiumhyaluronate.

In preferred embodiments, the buffering agent is histidine in aconcentration of about 10 mM to about 40 mM such as about 10 mM, 20 mM,30 mM, or 40 mM. In other preferred embodiments, the histidine is in aconcentration of about 20 mM to about 30 mM such as about 20 mM, 21 mM,22 mM, 23 mM, 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, or 30 mM. Inother preferred embodiments, the histidine is in a concentration ofabout 25 mM.

In particular embodiments, the pharmaceutical formulation comprises asurfactant. In preferred embodiments, the surfactant is in aconcentration of about 0.01 to about 4% w/v such as about 0.01%, 0.5%,1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, or 4.0%. In other preferredembodiments, the surfactant is in a concentration of about 0.01% toabout 1.0% w/v such as about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%,0.07%, 0.08%, 0.09%, 1.0% w/v. In other preferred embodiments thesurfactant is in a concentration of about 0.03% w/v.

In preferred embodiments, the surfactant is selected from a groupconsisting of polysorbate 20, polysorbate 80 and poloxamer 188,preferably polysorbate 20. In preferred embodiments, polysorbate 20 isin a concentration of about 0.03% w/v.

In some embodiments, the fusion protein is in a pharmaceuticalformulation that is stable at −70° C. to 5° C. for at least two yearssuch as −70° C., −60° C., −50° C., −40° C., −30° C., −20° C., −10° C.,0° C., or 5° C. In some embodiments, the fusion protein is in apharmaceutical formulation that is stable at −70° C., −20° C., and/or 5°C. for at least 6 months such as 6 months, 7 months, 8 months, 9 months,10 months, 11 months, or more. In preferred embodiments, the fusionprotein is in a pharmaceutical formulation that is stable at −70° C.,−20° C., and/or 5° C. for at least one year, two years, three years,four years, five years, six years, seven years, eight years, nine years,or ten years or more. In other preferred embodiments, the fusion proteinis in a pharmaceutical formulation that is stable at −70° C. −20° C.,and/or 5° C. for at least two years.

In some embodiments, the formulation retains protein purity and potencyafter at least 6 months at −70° C. to 25° C. such as −70° C., −60° C.,−50° C., −40° C., −30° C., −20° C., −10° C., 0° C., or 5° C. or 25° C.In some embodiments, the formulation retains protein purity and potencyafter at least 6 months at −70° C., −20° C., and/or 5° C. such as 6months, 7 months, 8 months, 9 months, 10 months, 11 months, one year,two years, three years, four years, five years, six years, seven years,eight years, nine years, or ten years or more.

In some embodiments, the formulation further comprises a salt. In someembodiments, the salt is selected from sodium chloride, magnesiumchloride, calcium chloride, or potassium chloride.

In particular embodiments, the formulation further comprises at leastone amino acid. In some embodiments, the amino acid is selected from thegroup consisting of arginine, histidine, methionine, proline, cysteine,lysine, glycine, aspartate, tryptophan, glutamate, and isoleucine.

In some embodiments, the pharmaceutical formulation comprises a fusionprotein in a concentration of 40 mg/mL, 25 mM histidine, 190 mMtrehalose, and 0.03% polysorbate 20, wherein the formulation is at a pHof about 6.0.

The present invention also provides a method for making any formulationset forth herein comprising the step of combining each component of theformulation into a single composition. Such a method may include thestep of adding the resulting formulation into a vial or injectiondevice. The method may additionally include a sterile filtration step.Any composition that is the product of such a method also forms part ofthe present invention. For example, embodiments herein also includemethods for preparing a formulation by combining a histidine-basedbuffer with a surfactant (such as polysorbate 20), a fusion protein,trehalose, and optionally, one or more additional components, e.g., asdiscussed herein.

The present invention also relates to a use of the composition accordingto the present invention to treat or prevent an integrin-associateddisease in an individual or a subject. An “individual” or “subject” is amammal. Mammals include, but are not limited to, domesticated animals(e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans andnon-human primates such as monkeys), rabbits and rodents (e.g., mice andrats). In some embodiments, a method of treating or preventing one ormore aspects or symptoms of a disease comprises administering aneffective amount of a composition comprising the fusion protein to anindividual.

The methods described herein can be used for the treatment of a varietyof diseases, including but not limited to, inflammatory disease, oculardisease, autoimmune disease, or cancer. In some embodiments, the diseaseto be treated includes, but is not limited to, rheumatoid arthritis,inflammatory arthritis, osteoarthritis, cancer, tissue/organ fibrosis,retinitis pigmentosa, uveitis (such as anterior uveitis or posterioruveitis) and ocular disease characterized by neovascularization orischemia (such as corneal neovascularization, iris neovascularization,neovascularization glaucoma, post-surgical fibrosis in glaucoma,proliferative vitreoretinopathy (PVR), choroidal neovascularization(CNV), optic disc neovascularization, retinal neovascularization,vitreal neovascularization, pannus, pterygium, vascular retinopathy,diabetic retinopathy (DR, non-proliferative and proliferative DR)without DME, diabetic retinopathy (DR, non-proliferative andproliferative DR) with DME, diabetic macular edema (DME), exudative(wet) and non-exudative (dry) age-related macular degeneration (AMD),macular edema, macular edema following retinal vein occlusion (RVO),retinal vein occlusion (RVO), central retinal vein occlusion (CRVO),Branch retinal vein occlusion (BRVO), Retinal Angiomatous Proliferation(RAP), polypoidal choroidal vascularization (PCV)), vitreomacularadhesion (VMA) and/or vitreomacular traction (VMT).

The compositions described herein can be administered to an individualvia any route, including, but not limited to, intravenous,intraperitoneal, ocular, intra-arterial, intrapulmonary, oral,inhalation, intravesicular, intramuscular, intratracheal, subcutaneous,intrathecal, transdermal, transpleural, topical, mucosal,gastrointestinal, intraarticular, intracisternal, intraventricular,intracranial, intraurethral, intrahepatic and intratumoral. In someembodiments, the compositions are administered systemically (for exampleby intravenous injection). In some embodiments, the compositions areadministered locally (for example by intraarterial or intraocularrelated injection thereof).

In some embodiments, the compositions are administered directly to theeye or the eye tissue. In some embodiments, the compositions areadministered topically to the eye, for example, in eye drops. In someembodiments, the compositions are administered by injection to the eyeor to the tissues associated with the eye. The compositions can beadministered, for example, by intraocular injection, periocularinjection, subretinal injection, intravitreal injection, superchoroidalinjection, trans-septal injection, subscleral injection, intrachoroidalinjection, intracameral injection, subconjunctival injection,sub-Tenon's injection, retrobulbar injection, peribulbar injection, orposterior juxtascleral delivery. These methods are known in the art. Thecompositions may be administered, for example, to the vitreous, aqueoushumor, sclera, conjunctiva, the area between the sclera and conjunctiva,the retina choroids tissues, macula, or other area in or proximate tothe eye of an individual.

In some embodiments, the pharmaceutical composition is administered tothe eye at a dose of about 0.03-10 mg per eye, such as 0.03 mg, 0.04 mg,0.05 mg, 0.06 mg, 0.07 mg, 0.08 mg, 0.09 mg, 0.1 mg, 0.2 mg, 0.3 mg, 0.4mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 mg, 2 mg, 3 mg, 4 mg, 5mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. In preferred embodiments, thepharmaceutical composition is delivered to the eye at a dose of about3.0-6.0 mg per eye such as 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 5.5mg, or 6 mg. In other preferred embodiments, the pharmaceuticalcomposition is delivered to the eye at a dose of about 4 mg per eye.

The optimal effective amount of the compositions can be determinedempirically and will depend on the type and severity of the disease,route of administration, disease progression and health, mass and bodyarea of the individual. Such determinations are within the skill of onein the art. Compositions comprising a fusion protein can also beadministered six times a week, five times a week, four times a week,three times a week, twice a week, once a week, once every two weeks,once every three weeks, once a month, once every two months, once everythree months, once every four months, once every six months, once everynine months, or once every year.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to describe the methods and/ormaterials in connection with which the publications are cited.

Embodiments

The application includes, but is not limited to, the following numberedembodiments:

Embodiment 1 is a pharmaceutical formulation, the formulationcomprising:

-   -   a) a fusion protein in a concentration of about 0.5 mg/mL to        about 120 mg/mL,    -   b) a polyol or alcohol selected from a group consisting of        sucrose, trehalose, mannitol, sorbitol, benzyl alcohol,        polyvinyl alcohol, polyethylene glycol (PEG) 400-12000, in a        concentration of about 1% to about 10% w/v,    -   c) a buffering agent selected from a group consisting of sodium        phosphate, histidine, sodium citrate, sodium acetate, sodium        bicarbonate, and trisodium citrate dihydrate in a concentration        of about 10 mM to about 50 mM, and    -   d) a surfactant in a concentration of about 0.01 to about 4%        w/v,        wherein the formulation is at a pH of about 5.5-7.5 and        optionally, the formulation further comprises a polysaccharide        selected from the group consisting of sodium        carboxymethylcellulose, microcrystalline cellulose, or sodium        hyaluronate.

Embodiment 2 is the pharmaceutical formulation of embodiment 1, whereinthe surfactant is selected from a group consisting of polysorbate 20,polysorbate 80 and poloxamer 188, preferably polysorbate 20.

Embodiment 3 is the pharmaceutical formulation of embodiment 2, whereinthe polysorbate is polysorbate 20.

Embodiment 4 is the pharmaceutical formulation of any one of embodiments1-3, wherein the surfactant is in a concentration of about 0.03% w/v.

Embodiment 5 is the pharmaceutical formulation of any one of embodiments1-4, wherein the fusion protein is in a concentration of about 1 mg/mLto about 90 mg/mL, preferably about 20 mg/mL to about 80 mg/mL, morepreferably the fusion protein is in a concentration of about 40 mg/mL.

Embodiment 6 is the pharmaceutical formulation of embodiment 5, whereinthe fusion protein is in a concentration of about 40 mg/mL.

Embodiment 7 is the pharmaceutical formulation of any one of embodiments1-6, wherein the polyol is trehalose in a concentration of about 25 mMto about 250 mM, preferably about 190 mM.

Embodiment 8 is the pharmaceutical formulation of embodiment 7, whereintrehalose is in a concentration of about 190 mM.

Embodiment 9 is the pharmaceutical formulation of any one of embodiments1-8, wherein the buffering agent is histidine in a concentration ofabout 10 mM to about 40 mM, preferably about 20 mM to about 30 mM, morepreferably the histidine is in a concentration of about 25 mM.

Embodiment 10 is the pharmaceutical formulation of embodiment 9, whereinthe histidine is in a concentration of about 25 mM.

Embodiment 11 is the pharmaceutical formulation of any one ofembodiments 1-10, wherein the fusion protein comprises, from N-terminusto C-terminus in the following order:

-   -   a) an extracellular domain of a Vascular Endothelial Growth        Factor receptor (VEGFR);    -   b) an Fc domain of human immunoglobulin G; and    -   c) an integrin binding protein or its fragment thereof.

Embodiment 12 is the pharmaceutical formulation of any one ofembodiments 1-11, wherein the fusion protein comprises, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO: 17, or SEQ ID NO: 18.

Embodiment 13 is the pharmaceutical formulation of any one ofembodiments 1-12, wherein the pH is about 5.5 to about 7.0, preferablythe pH is about 6.0.

Embodiment 14 is the pharmaceutical formulation of embodiment 13,wherein the pH is about 6.0.

Embodiment 15 is the pharmaceutical formulation of any one ofembodiments 1-14, wherein the formulation is stable at −70° C., −20° C.and/or 2-8° C. for at least 24 months.

Embodiment 16 is the pharmaceutical formulation of any one ofembodiments 1-15, wherein the formulation retains protein purity andpotency after least 6 months at −70° C., −20° C. and/or 2-8° C.,preferably at 2-8° C.

Embodiment 17 is the pharmaceutical formulation of any one ofembodiments 1-16, wherein the formulation further comprises a salt in aconcentration of about 10 mM to 50 mM.

Embodiment 18 is the pharmaceutical formulation of embodiment 17,wherein the salt is selected from sodium chloride, magnesium chloride,calcium chloride, or potassium chloride.

Embodiment 19 is the pharmaceutical formulation of any one ofembodiments 1-18, further comprises at least one amino acid in aconcentration of about 10 mM to 50 mM.

Embodiment 20 is the pharmaceutical formulation of embodiment 19,wherein the amino acid is selected from the group consisting ofarginine, methionine, proline, histidine, cysteine, lysine, glycine,aspartate, tryptophan, glutamate, and isoleucine.

Embodiment 21 is the pharmaceutical formulation of any one ofembodiments 1-20, wherein the formulation is used for a method oftreating an ocular disease.

Embodiment 22 is the pharmaceutical formulation of embodiment 21,wherein the ocular disease is selected from neovascularization orischemia uveitis, retinal vasculitis, angioid streaks, retinitispigmentosa, corneal neovascularization, iris neovascularization,neovascularization glaucoma, post-surgical fibrosis in glaucoma,proliferative vitreoretinopathy (PVR), choroidal neovascularization(CNV), optic disc neovascularization, retinal neovascularization,vitreal neovascularization, pannus, pterygium, vascular retinopathy,diabetic retinopathy (DR, non-proliferative and proliferative DR)without DME, diabetic retinopathy (DR, non-proliferative andproliferative DR) with DME, diabetic macular edema (DME), exudative(wet) and non-exudative (dry) age-related macular degeneration (AMD),macular edema, macular edema following retinal vein occlusion (RVO),retinal vein occlusion (RVO), central retinal vein occlusion (CRVO),Branch retinal vein occlusion (BRVO), Retinal Angiomatous Proliferation(RAP), polypoidal choroidal vascularization (PCV), vitreomacularadhesion (VMA) and/or vitreomacular traction (VMT).

Embodiment 23 is the pharmaceutical formulation of any one ofembodiments 1-22, wherein the formulation is administered at a dose ofabout 0.03-10 mg per eye, preferably about 3.0-6.0 mg per eye, morepreferably the formulation is administered at a dose of about 4 mg pereye.

Embodiment 24 is the pharmaceutical formulation of embodiment 23,wherein the formulation is administered at dose of about 4 mg per eye.

Embodiment 25 is a pharmaceutical formulation, the formulationcomprising:

-   -   a) a fusion protein in a concentration of 40 mg/mL,    -   b) 25 mM histidine,    -   c) 190 mM trehalose, sucrose, or mannitol,    -   d) 0.03% polysorbate 20, or polysorbate 80,        wherein the formulation is at a pH of about 6.0.

Embodiment 26 is the pharmaceutical formulation of embodiment 25,wherein the fusion protein comprises, from N-terminus to C-terminus inthe following order:

-   -   a) an extracellular domain of a Vascular Endothelial Growth        Factor receptor (VEGFR);    -   b) an Fc Domain of human immunoglobulin G;    -   c) an integrin binding protein or its fragment thereof.

Embodiment 27 is the pharmaceutical formulation of embodiment 26,wherein the fusion protein comprises SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO: 17, or SEQ ID NO: 18.

Embodiment 28 is the pharmaceutical formulation of any one ofembodiments 25-27, wherein the formulation is stable at −70° C., −20° C.and/or 5° C. for at least 24 months.

Embodiment 29 is the pharmaceutical formulation of any one ofembodiments 25-28, wherein the formulation retains protein purity andpotency after at least 6 months at −70° C., −20° C. and/or 2-8° C.,preferably at 2-8° C.

Embodiment 30 is the pharmaceutical formulation of any one ofembodiments 25-29, wherein the formulation further comprises a salt in aconcentration of about 10 mM to 50 mM.

Embodiment 31 is the pharmaceutical formulation of embodiment 30,wherein the salt is selected from sodium chloride, magnesium chloride,calcium chloride, or potassium chloride.

Embodiment 32 is the pharmaceutical formulation of any one ofembodiments 25-31, wherein the formulation further comprises at leastone amino acid in a concentration of about 10 mM to 50 mM.

Embodiment 33 is the pharmaceutical formulation of embodiment 32,wherein the amino acid is selected from the group consisting ofarginine, methionine, proline, histidine, cysteine, lysine, glycine,aspartate, tryptophan, glutamate, and isoleucine.

Embodiment 34 is the pharmaceutical formulation of any one ofembodiments 25-33, wherein the formulation is used for a method oftreating an ocular disease.

Embodiment 35 is the pharmaceutical formulation of embodiment 34,wherein the ocular disease is selected from neovascularization orischemia uveitis, retinal vasculitis, angioid streaks, retinitispigmentosa, corneal neovascularization, iris neovascularization,neovascularization glaucoma, post-surgical fibrosis in glaucoma,proliferative vitreoretinopathy (PVR), choroidal neovascularization(CNV), optic disc neovascularization, retinal neovascularization,vitreal neovascularization, pannus, pterygium, vascular retinopathy,diabetic retinopathy (DR, non-proliferative and proliferative DR)without DME, diabetic retinopathy (DR, non-proliferative andproliferative DR) with DME, diabetic macular edema (DME), exudative(wet) and non-exudative (dry) age-related macular degeneration (AMD),macular edema, macular edema following retinal vein occlusion (RVO),retinal vein occlusion (RVO), central retinal vein occlusion (CRVO),Branch retinal vein occlusion (BRVO), Retinal Angiomatous Proliferation(RAP), polypoidal choroidal vascularization (PCV), vitreomacularadhesion (VMA) and/or vitreomacular traction (VMT).

Embodiment 36 is the pharmaceutical formulation of any one ofembodiments 25-35, wherein the formulation is administered at a dose ofabout 0.03-10 mg per eye, preferably about 3.0-6.0 mg per eye, morepreferably the formulation is administered at a dose of about 4 mg pereye.

Embodiment 37 is the pharmaceutical formulation of embodiment 36,wherein the formulation is administered at dose of about 4 mg per eye.

Embodiment 38 is a method of treating an ocular disease in a subject,the method comprising administering to the subject a pharmaceuticalformulation comprising:

-   -   a) a fusion protein in a concentration of about 0.5 mg/mL to        about 120 mg/mL,    -   b) a polyol or alcohol selected from a group consisting of such        as sucrose, trehalose, mannitol, sorbitol, benzyl alcohol,        polyvinyl alcohol, PEG 400-12000 in a concentration of about 1%        to about 10% w/v,    -   c) a buffering agent selected from a group consisting of sodium        phosphate, histidine, sodium citrate, sodium acetate, sodium        bicarbonate, and trisodium citrate dihydrate in a concentration        of about 10 mM to about 50 mM,    -   d) a surfactant in a concentration of about 0.01 to about 4%        w/v,        wherein the pH is about 5.5-7.5 and optionally, the formulation        further comprises a polysaccharide selected from the group        consisting of sodium carboxymethylcellulose, microcrystalline        cellulose, or sodium hyaluronate.

Embodiment 39 is the method of embodiment 38, wherein the surfactant isselected from a group consisting of polysorbate 20, polysorbate 80 andpoloxamer 188, preferably polysorbate 20.

Embodiment 40 is the method of embodiment 39, wherein the surfactant ispolysorbate 20.

Embodiment 41 is the method of any one of embodiments 38-40, wherein thesurfactant is in a concentration of about 0.03% w/v.

Embodiment 42 is the method of any one of embodiments 38-41, wherein thefusion protein is in a concentration of about 1 mg/mL to about 90 mg/mL,preferably about 40 mg/mL to about 80 mg/mL, more preferably the fusionprotein is a concentration of about 40 mg/mL.

Embodiment 43 is the method of embodiment 42, wherein the fusion proteinis in a concentration of about 40 mg/mL.

Embodiment 44 is the method of any one of embodiments 38-43, wherein thepolyol is trehalose in a concentration of about 150 mM to about 230 mM,preferably about 190 mM.

Embodiment 45 is the method of embodiment 44, wherein trehalose is in aconcentration of about 190 mM.

Embodiment 46 is the method of any one of embodiments 38-45, wherein thebuffer is histidine in a concentration of about 20 mM to about 40 mM,preferably about 20 mM to about 30 mM, more preferably the histidine isin a concentration of about 25 mM.

Embodiment 47 is the method of embodiment 46, wherein the histidine isin a concentration of about 25 mM.

Embodiment 48 is the method of any one of embodiments 38-47, wherein thefusion protein comprises, from N-terminus to C-terminus in the followingorder:

-   -   a) an extracellular domain of a Vascular Endothelial Growth        Factor receptor (VEGFR);    -   b) an Fc domain of human immunoglobulin G; and    -   c) an integrin binding protein or its fragment thereof.

Embodiment 49 is the method of any one of embodiments 38-48, wherein thefusion protein comprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, orSEQ ID NO: 18.

Embodiment 50 is the method of any one of embodiments 38-49, wherein thepH is about 6.0 to about 6.5, preferably the pH is about 6.0.

Embodiment 51 is the method of embodiment 50, wherein the pH is about6.0.

Embodiment 52 is the method of any one of embodiments 38-51, wherein theformulation is stable at −70° C., −20° C. and/or 2-8° C. for at least 24months.

Embodiment 53 is the method of any one of embodiments 38-52, wherein theformulation retains protein purity and potency after at least 6 monthsat −70° C., −20° C. and/or 2-8° C., preferably at 2-8° C.

Embodiment 54 is the method of any one of embodiments 38-53, whereinformulation further comprises a salt in a concentration of about 10 mMto 50 mM.

Embodiment 55 is the method of embodiment 54, wherein the salt isselected from sodium chloride, magnesium chloride, calcium chloride, orpotassium chloride.

Embodiment 56 is the method of any one of embodiments 38-55, wherein theformulation further comprises at least one amino acid in a concentrationof about 10 mM to 50 mM.

Embodiment 57 is the method of embodiment 56, wherein the amino acid isselected from the group consisting of arginine, methionine, proline,histidine, cysteine, lysine, glycine, aspartate, tryptophan, glutamate,and isoleucine.

Embodiment 58 is the method of any one of embodiments 38-57, wherein theocular disease comprises neovascularization or ischemia uveitis, retinalvasculitis, retinitis pigmentosa, angioid streaks, cornealneovascularization, iris neovascularization, neovascularizationglaucoma, post-surgical fibrosis in glaucoma, proliferativevitreoretinopathy (PVR), choroidal neovascularization (CNV), optic discneovascularization, retinal neovascularization, vitrealneovascularization, pannus, pterygium, vascular retinopathy, diabeticretinopathy (DR, non-proliferative and proliferative DR) without DME,diabetic retinopathy (DR, non-proliferative and proliferative DR) withDME, diabetic macular edema (DME), exudative (wet) and non-exudative(dry) age-related macular degeneration (AMD), macular edema, macularedema following retinal vein occlusion (RVO), retinal vein occlusion(RVO), central retinal vein occlusion (CRVO), Branch retinal veinocclusion (BRVO), Retinal Angiomatous Proliferation (RAP), polypoidalchoroidal vascularization (PCV), vitreomacular adhesion (VMA) and/orvitreomacular traction (VMT).

Embodiment 59 is the method of any one of embodiments 38-58, wherein theformulation is administered at a dose of about 0.03-10 mg per eye,preferably about 3.0-6.0 mg per eye, more preferably the formulation isadministered at a dose of about 4 mg per eye.

Embodiment 60 is the method of embodiment 59, wherein the formulation isadministered at dose of about 4 mg per eye.

Embodiment 61 is a method of treating an ocular disease in a subject,the method comprising administering to the subject a pharmaceuticalformulation comprising:

-   -   a) a fusion protein in a concentration of 40 mg/mL,    -   b) 25 mM histidine,    -   c) 190 mM trehalose, sucrose, or mannitol,    -   d) 0.03% polysorbate 20, or polysorbate 80, wherein the        formulation is at a pH of about 6.0.

Embodiment 62 is the method of embodiment 61, wherein the fusion proteincomprises, from N-terminus to C-terminus in the following order:

-   -   a) an extracellular domain of a Vascular Endothelial Growth        Factor receptor (VEGFR);    -   b) an Fc Domain of human immunoglobulin G;    -   c) an integrin binding protein or its fragment thereof.

Embodiment 63 is the method of embodiment 62, wherein the fusion proteincomprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, or SEQ ID NO: 18.

Embodiment 64 is the method of any one of embodiments 61-63, wherein theformulation is stable at −70° C., −20° C. and/or 5° C. for at least 24months.

Embodiment 65 is the method of any one of embodiments 61-64, wherein theformulation retains protein purity and potency after at least 6 monthsat −70° C., −20° C. and/or 2-8° C., preferably at 2-8° C.

Embodiment 66 is the method of any one of embodiments 61-65, wherein theformulation further comprises a salt in a concentration of about 10 mMto 50 mM.

Embodiment 67 is the method of embodiment 66, wherein the salt isselected from sodium chloride, magnesium chloride, calcium chloride, orpotassium chloride.

Embodiment 68 is the method of any one of embodiments 61-67, wherein theformulation further comprises at least one amino acid in a concentrationof about 10 mM to 50 mM.

Embodiment 69 is the method of embodiment 68, wherein the amino acid isselected from the group consisting of arginine, methionine, proline,histidine, cysteine, lysine, glycine, aspartate, tryptophan, glutamate,and isoleucine.

Embodiment 70 is the method of any one of embodiments 61-69, wherein theocular disease is selected from neovascularization or ischemia uveitis,retinal vasculitis, angioid streaks, retinitis pigmentosa, cornealneovascularization, iris neovascularization, neovascularizationglaucoma, post-surgical fibrosis in glaucoma, proliferativevitreoretinopathy (PVR), choroidal neovascularization (CNV), optic discneovascularization, retinal neovascularization, vitrealneovascularization, pannus, pterygium, vascular retinopathy, diabeticretinopathy (DR, non-proliferative and proliferative DR) without DME,diabetic retinopathy (DR, non-proliferative and proliferative DR) withDME, diabetic macular edema (DME), exudative (wet) and non-exudative(dry) age-related macular degeneration (AMD), macular edema, macularedema following retinal vein occlusion (RVO), retinal vein occlusion(RVO), central retinal vein occlusion (CRVO), Branch retinal veinocclusion (BRVO), Retinal Angiomatous Proliferation (RAP), polypoidalchoroidal vascularization (PCV), vitreomacular adhesion (VMA) and/orvitreomacular traction (VMT).

Embodiment 71 is the method of any one of embodiments 61-70, wherein theformulation is administered at a dose of about 0.03-10 mg per eye,preferably about 3.0-6.0 mg per eye, more preferably the formulation isadministered at a dose of about 4 mg per eye.

Embodiment 72 is the method of embodiment 71, wherein the formulation isadministered at dose of about 4 mg per eye.

EXAMPLES

The following examples are offered to illustrate but not to limit theinvention. One of skill in the art will recognize that the followingprocedures may be modified using methods known to one of ordinary skillin the art.

Example 1. Evaluation of Fusion Protein 1 in a Laser-Induced ChoroidalNeovascularization Model in Rhesus Monkeys

Fusion Protein 1 (an IgG1 Fc-fusion protein with multiple targets) wasevaluated in a monkey model of choroidal neovascularization (CNV) in theeyes.

In the CNV model, rhesus monkeys (n=4 eyes per group) received laserburns on the retina using photocoagulation, and choroidalneovascularization was allowed to develop for 21 days (Day −21 to Day0). On Day 0, Vehicle, 0.5 mg/eye Lucentis® (0.5 mg/eye) or FusionProtein 1 (0.6 mg, 1.0 mg, 1.9 mg/eye) were injected into the monkeyeyes via a single intravitreal (IVT) injection. Efficacy endpoints wereevaluated on baseline (Day −2) prior to dosing, and on Day 14 and Day 28post-injection in all groups.

In the eyes treated with 0.5 mg Lucentis®, mean leakage area scoresdecreased by 91% and 92% on Day 14 and 28, respectively, compared tobaseline (FIG. 1 ). Mean retinal thickness also decreased by 117% and120%, respectively, compared to baseline (FIG. 2 ). Lucentis® showedstatistically significant improvement compared to eyes treated with thevehicle control.

All three dose groups of Fusion Protein 1 exhibited recovery of thelaser-induced retinal thickness, and leakage area compared to baselineand pre-study values. Mean leakage area scores decreased by 87% to 89and 79% to 90% on Day 14 and 28, respectively (FIG. 1 ). Mean retinalthickness also decreased by 98% to 121% and 104% to 129%, respectively(FIG. 2 ). Fusion Protein 1 showed statistically significant improvementcompared to eyes treated with the vehicle control on Day 14 and Day 28for the 0.6 mg and 1.0 mg doses. There was no dose-dependent effect ofFusion Protein 1 observed, as all doses showed similar efficacy.

Histology examinations of eyes using Masson's staining for collagen (aprecursor to fibrosis) showed a dose-dependent reduction in collagenthickness (ocular fibrosis) for Fusion Protein 1, but not for vehicle orLucentis® treated groups (FIG. 3 ).

In conclusion, a single IVT injection of Fusion Protein 1 at 0.6, 1.0,and 1.9 mg/eye effectively inhibited vascular leakage, retinalthickness, and fibrosis in a laser-induced CNV model in rhesus monkeys.Based on the vitreal volume ratio between human (4 mL) and monkey (2mL), these doses equate to a human equivalent dose of 1.2 mg, 2 mg, and4 mg/eye. This supports the proposed doses of Fusion Protein 1 in thetreatment of retinal diseases such as diabetic macular edema (DME) andneovascular age-related macular degeneration (nAMD). Furthermore, thedata suggests that Fusion Protein 1 has an additive effect to benefitpatients compared to the conventional anti-VEGF monotherapy.

Example 2. Evaluation of Fusion Protein 1 in a Bleomycin-Induced C57BL/6Mouse Lung Fibrosis Model

Fusion Protein 1, administered intravenously (IV) at 39 mg/kg, wasevaluated for possible anti-fibrotic activity in a bleomycin-inducedlung fibrosis mouse model. The mice were challenged with bleomycin at1.5 U/kg by using a PENNCENTURY™ intrapulmonary aerosolizer on Day 1.The mice were intravenously administered Fusion Protein 1, NintedanibEthanesulfonate (Nintedanib) or vehicle (formulation buffer), daily fromDay 1 to Day 21, and were sacrificed on Day 22. The total protein wasprecipitated out from collected lung tissue with 50% trichloroaceticacid on ice for 20 minutes. Samples were centrifuged and the pellet wasmixed with 1 mL 12 N HCl and baked at 110° C. for 14-18 hours untilsamples were charred and dry. The samples were resuspended in 2 mLdeionized water by incubating for 72 hours at room temperature whileapplying intermittent vortexing. Serial dilutions oftrans-4-hydroxy-L-proline standard (source: Sigma, USA) were preparedstarting at 0.5 mg/mL. The 200 μL vortexed sample (or standard) wasadded to 500 μL 1.4% chloramine T in 0.5 M sodium acetate/10%isopropanol (source: Fisher Sci, USA) and incubated for 20 minutes atroom temperature. Next, 500 μL Ehrlich's solution (1.0 Mp-dimethylaminobenzaldehyde in 70% isopropanol/30% perchloric acid)(source: Fisher Sci, USA) was added, mixed, and incubated at 65° C. for15 minutes. After samples reached room temperature, the optical densityof each sample and standard was measured at 550 nm and the concentrationof lung hydroxyproline (μg/lung) was calculated from the hydroxyprolinestandard curve. Proline is a major component of collagen, which is oneof the markers used to assess fibrosis formation.

Lung hydroxyproline was significantly increased (p<0.05) in the vehiclecontrol group (FIG. 4 ), suggesting a successful induction of lungfibrosis. Multiple administrations of Fusion Protein 1 markedlyinhibited the hydroxyproline level (p<0.05) compared to the vehiclecontrol, signifying an additive effect on these markers following FusionProtein 1 injections (FIG. 4 ). Consecutive oral exposures of Nintedanibhad a mild effect on reducing the level of hydroxyproline (FIG. 4 ).

Thus, Fusion Protein 1 administrated intravenously at 39 mg/kg oncedaily for 21 days was associated with significant reduction in lunghydroxyproline, a major lung fibrosis marker of bleomycin-induced lungfibrosis in mice.

Example 3. Pre-Formulation of Fusion Protein 1 for IntravitrealInjection

A pre-formulation study of Fusion Protein 1 was performed to develop asuitable liquid formulation of Fusion Protein 1. The objectives of thestudy were as follows:

-   -   1. To evaluate compatibility and colloidal stability of various        concentrations Fusion Protein 1 (40 mg/mL and 80 mg/mL) with        selected buffers,    -   2. To evaluate the changes of quality attributes under        accelerated conditions (40° C., 25° C., 2-8° C.) after        short-term storage,    -   3. Excipient selection: Tonicity, NaCl, polyols (i.e.,        trehalose, sucrose, mannitol, and sorbitol) and other        stabilizers such as methionine, and arginine,    -   4. Evaluate compatibility through design of experiment (DoE)        plan with the selected buffer and potential stabilizers; and    -   5. Assess the effect of cryoprotectants

The formulation development work on the drug substance was initiatedusing a DoE approach to identify appropriate buffer, polyols,surfactants, and other stabilizers. Two concentrations, 40 and 80 mg/mLof Fusion Protein 1, were tested during the formulation development.Thermal stress as well as agitation and freeze/thaw stress were employedduring the formulation development. Table 2 summarizes the ingredientsand associated function tested in the following studies.

TABLE 2 Investigational formulation summary (ingredients screened).Tested Items Ingredients Function Concentration Buffer Citric AcidBuffering agent 25 mM, pH 5.5 & pH 6.0 Histidine 25 mM, pH 6.0 & pH 6.5Sodium Phosphate 25 mM, pH 6.5 & pH 7.0 Amino Methionine Buffering agent 40 mM acid Arginine  40 mM Salt NaCl Tonicity and Buffer  40 mM agentPolyols Sucrose Tonicity and 190 mM Mannitol Stabilizer Agent 190 mMTrehalose 190 mM Sorbitol 190 mM Surfactant PS20 Stabilizer Agent 0.03%PS80 0.03%

Colloidal Stability and Compatibly of Fusion Protein 1 in VariousBuffers and pHs

The solubility and diffusion coefficient (D) is commonly used toevaluate protein-protein interactions and is also used to demonstrateprotein colloidal stability. Colloidal stability is a critical parameterto represent the long-term integrity of a molecule after dispersion andits resistance to sedimentation/precipitation in the solution. Inaddition, this method can rapidly capture the compatibility of a buffertoward the molecule. Consequently, Fusion Protein 1 was subjected tothree commonly used ophthalmic solution buffers, sodium phosphate(NaPi), histidine (His), and citrate buffer (Cit) with potentialbuffering pH (Table 3). The attributes of Fusion Protein 1 were analyzedby size exclusion chromatography of high-performance liquidchromatography (SEC-HPLC) (Table 4), dynamic light scattering (DLS)(Table 5), and turbidity (FIG. 6 ).

TABLE 3 Investigated buffer systems and corresponding pH range. BufferTest Buffer pH 25 mM Sodium phosphate (Phosphate buffer) 7.0 6.5 25 mML-Histidine/L-Histidine hydrochloride 6.5 6.0 (Histidine buffer) 25 mMCitric Acid/Sodium Citrate (Citrate buffer) 6.0 5.5

The diffusion coefficient of Fusion Protein 1 in phosphate and citratebuffers exhibited negative correlation with increasing concentration,which indicated the possibility of increased protein-protein interaction(FIGS. 5A, 5B, 5E, and 5F). In contrast, a repulsive protein-proteininteraction of Fusion Protein 1 in histidine buffer at both 6.5 and 6.0was observed with a positive correlation between diffusion coefficientand various concentrations of Fusion Protein 1 (FIGS. 5C and 5D). Thus,a repulsive intermolecular interaction Fusion Protein 1 in histidinebuffer suggests favorable colloidal stability of Fusion Protein 1.

Descending diffusion coefficient was detected for all formulations, evenfor the histidine buffer formulations, as the concentration wasincreased above 40 mg/mL to 60 and 80 mg/mL Fusion Protein 1. Forhistidine buffer formulations, this suggests that preliminaryaggregation may occur once the concentration is increased above 40mg/mL. Based on results of SEC-HPLC, protein formulated at 80 mg/mLshowed lower purity in buffer and greater high molecular weightaggregate for all buffers and pH, with the exception of histidinebuffer, pH 6.5 (Table 4). Increased subvisible particles (100-1,000 nm,by DLS) were detected in all 80 mg/mL formulations compared to samplesat 40 mg/mL (Table 5). There was no significant difference in turbiditybetween 40 and 80 mg/mL samples (FIGS. 6A and 6B). Taken together,particle size analysis by DLS showed an increase in subvisibleaggregates for all buffers as the protein concentration was increasedfrom 40 to 80 mg/mL, a slight increase in HMW by SEC-HPLC was detectedfor all formulations except histidine buffer, pH 6.5, and no significantincrease in turbidity was observed for any of the formulations.

TABLE 4 SEC-HPLC purity and UV concentration results of Fusion Protein 1in different buffers. Concentration (mg/mL) SEC-HPLC (%) by A280 MainSample Planned Tested HMW^(a) peak^(b) Reference 1^(#) 40 37.7 0.6699.34 25 mM Citrate buffer, 40 45.2 0.55 99.45 pH 5.5 80 76.9 0.77 99.2325 mM Citrate buffer, 40 43.0 0.61 99.39 pH 6.0 80 73.8 0.79 99.21 25 mMHistidine buffer, 40 42.8 0.48 99.52 pH 6.0 80 79.7 0.52 99.48 25 mMHistidine buffer, 40 42.6 0.55 99.45 pH 6.5 80 74.1 0.44 99.56 25 mMPhosphate buffer, 40 38.5 0.60 99.40 pH 6.5 80 81.3 1.01 98.99 25 mMPhosphate buffer, 40 44.7 0.69 99.31 pH 7.0 80 83.4 0.98 99.02^(#)Reference 1 is formulated in 25 mM histidine, 6% sucrose, 20 mMNaCl, 0.03% PS20, pH 6.0. ^(a)HMW: aggregate as high molecule weightspecies with retention time at 8.3 ± 0.1 min; ^(b)Main peak retentiontime is 9.5 ± 0.1 min, which represents non-aggregated dimeric form ofFusion Protein 1.

TABLE 5 DLS sub-visible particle distribution in different buffers.Average radius of Average radius of Average radius of Conc. firstparticle second particle third particle Buffer pH (mg/mL) (0.1-10 nm) %Pd (10-100 nm) % Pd (100-1000 nm) % Pd Phosphate 7.0 40 8.86 22.50 — — —— 80 8.93 13.09 — — 364.12 10.88 6.5 40 7.85 15.14 — — — — 80 9.35 18.08— — 222.78 16.76 Histidine 6.5 40 4.86 54.30 — — — — 80 4.52 27.13 25.618.32 475.84 19.79 6.0 40 3.68 7.64 — — 213.06 6.28 80 3.40 5.40 52.198.50 799.23 11.84 Citrate 6.0 40 8.80 21.22 — — — — 80 9.78 17.64 — —322.11 11.93 5.5 40 8.60 16.05 — — — — 80 10.16 14.83 — — 305.76 13.83 %Pd = percent polydispersity

Buffer Selection and Fusion Protein 1 Attributes Assessment

The feasibility and accelerated stability of both 40 and 80 mg/mL FusionProtein 1 formulated in 25 mM citrate or histidine buffer at pH 6.0 weretested. Non-aggregated (dimeric form) quantity of Fusion Protein 1 incitrate and histidine buffer stored at 2-8° C. showed no obvious changeupon storage for both 40 and 80 mg/mL concentrations (Table 6). However,in accelerated condition at 40° C., the main peak for 80 mg/mL FusionProtein 1 decreased by approximately 70% in the citrate buffer after 28days (Table 7). In comparison, the content of Fusion Protein 1 decreasedby approximately 50% when formulated in histidine buffer. Furthermore,the protein concentration at 80 mg/mL Fusion Protein 1 decreased by 29%for citrate buffer compared to no decrease in the histidine buffer.Thus, Fusion Protein 1 in histidine buffer is more stable than incitrate buffer at pH 6.0.

TABLE 6 Changes of protein content, purity, and aggregate of FusionProtein 1 stored at 2-8° C. for 28 days. Planned Tested Concen- TimeConcen- SEC-HPLC (%) tration point tration Main Buffer, pH (mg/mL)(days) (mg/mL) HMW^(a) peak^(b) 25 mM 40 0 41.2 4.15 95.85 Citrate, 433.5 4.22 95.78 pH 6.0 7 36.0 4.43 95.57 14 36.3 4.46 95.54 28 35.5 4.6295.35 80 0 79.3 4.32 95.69 4 59.1 4.51 95.49 7 65.7 4.61 95.39 14 72.44.89 95.11 28 61.7 5.13 94.87 25 mM 40 0 38.5 4.07 95.93 Histidine, 435.3 4.28 95.72 pH 6.0 7 35.7 4.39 95.61 14 37.6 4.59 95.41 28 37.0 4.9295.08 80 0 78.5 4.22 95.79 4 51.3 4.47 95.53 7 53.1 4.73 95.27 14 63.85.00 95.00 28 60.5 5.40 94.60 Samples analyzed by UV absorbance at A₂₈₀(for concentration) and SEC-HPLC. ^(a)HMW: aggregate as high moleculeweight species with retention time at 8.3 ± 0.1 min; ^(b)Main peakretention time is 9.5 ± 0.1 min, which represents non-aggregated dimericform of Fusion Protein 1.

TABLE 7 Changes of protein content, purity, and aggregate of FusionProtein 1 at 40° C. for 28 days. Planned Time Tested SEC-HPLC (%) Conc.point Conc. Main Sample (mg/mL) (days) (mg/mL) HHMW^(#) HMW^(a) peak^(b)LMW* 25 mM 40 0 41.2 — 4.15 95.85 — Citrate, 4 33.9 10.02 14.94 75.04 —pH 6.0 7 30.3 15.60 20.25 63.02 1.13 14 37.4 28.78 21.34 47.67 2.21 2839.4 35.26 22.87 38.89 2.98 80 0 79.3 — 4.32 95.69 — 4 56.4 18.36 18.3062.63 0.71 7 62.2 28.11 20.03 50.56 1.30 14 69.3 40.47 21.45 36.11 1.9628 56.2 48.56 19.34 29.37 2.73 25 mM 40 0 38.5 — 4.07 95.93 — Histidine,4 35.3 1.82 7.84 90.34 — pH 6.0 7 35.7 1.44 11.04 87.53 — 14 38.9 4.6813.35 80.87 1.10 28 36.8 9.01 18.79 70.90 1.31 80 0 78.5 — 4.22 95.79 —4 61.7 3.85 11.22 84.93 — 7 67.8 5.73 14.42 79.33 0.52 14 81.2 11.3220.12 67.46 1.10 28 78.5 25.02 22.69 50.32 1.97 ^(a)HMW: aggregate ashigh molecule weight species with retention time at 8.3 ± 0.1 min^(b)Main peak retention time is 9.5 ± 0.1 min, which representsnon-aggregated dimeric form of Fusion Protein 1 ^(#)HHMW: aggregate ashigher molecule weight species which refer to a peak that has retentiontime at 7.6 ± 0.1 min *LMW: low molecular weight with retention time at13.5 ± 0.1 min “—”: Not detectable

Additionally, Fusion Protein 1 in citrate buffer showed greaterreduction of main band intensity at the position of approximately 250kDa in non-reducing SDS-PAGE (FIGS. 7A-7E) after incubation at 40° C.(FIGS. 8A-8E). The protein degradation profile of Fusion Protein 1resulted in increasing bands of product degraded fragments and highmolecular weight aggregate over time. Analysis by reducing SDS-PAGEshowed reduction of main band intensity at approximately 75 kDa andincreased fragment bands (FIGS. 8A-8E). Overall, protein purity andintegrity investigation indicated that Fusion Protein 1 exhibited betterstability in 25 mM histidine buffer at pH 6.0 than in citrate buffer.

The accelerated condition with short-term storage to freeze/thaw wasalso evaluated to examine changes of quality attributes of FusionProtein 1 samples. Samples were incubated at 40° C. for 4 days. Onefreeze thaw cycle of −70° C. and room temperature (RT) tests indicatedthat Fusion Protein 1 was stable without polyol protection (Table 8).

TABLE 8 Changes of protein purity and aggregate of Fusion Protein 1after one freeze-thaw cycle. Planned Conc. SEC-HPLC (%) Sample (mg/mL)Treatment HMW^(a) Main Peak^(b) 25 mM Citrate, 40 Before 4.15 95.85 pH6.0 After 4.13 95.76 80 Before 4.32 95.69 After 4.14 95.56 25 mMHistidine, 40 Before 4.07 95.93 pH 6.0 After 4.24 95.87 80 Before 4.2295.79 After 4.44 95.86 ^(a)HMW: aggregate as high molecule weightspecies with retention time at 8.3 ± 0.1 min ^(b)Main peak retentiontime is 9.5 ± 0.1 min, which represents non-aggregated dimeric form ofFusion Protein 1.

Excipient Screening

To investigate the suitability of excipients, a pilot study wasconducted to evaluate the main peak purity change by SEC-HPLC offormulated Fusion Protein 1 samples. Based on the studies describedabove, 25 mM histidine buffer pH 6.0 was selected for further excipientscreening tests. Three types of additives included were polyols, salt,and amino acid. Evaluations were designed to have eleven conditions andtested by accelerated condition at 40° C. for 4 days (Table 9).

TABLE 9 Excipients screened. Tested Stabilizer at 150 mM 40 mM Aminoacid at 40 mM #Test Trehalose Mannitol Sucrose Sorbitol NaCl ArginineMethionine 0 — — — — — — — 1 + — — — — — — 2 — + — — — — — 3 — — + — — —— 4 — — — + — — — 5 — — — — + — — 6 + — — — + — — 7 — + — — + — — 8 —— + — + — — 9 — — — + + — — 10 — — + — — + — 11 — — + — — — + Note:Fusion Protein 1 was formulated in 25 mM histidine buffer pH 6.0 andtested with each different polyol (test #1 to 4) or combined with 40 mMNaCl (test #6-9). Test# 5 = 40 mM NaCl without polyol or amino acid.Tests #10 and 11 = sucrose combined with 2 amino acids.

Main peak purity was ≥90% when formulated with each polyol (Table 10,tests #1-4), the combination of trehalose with NaCl (test #6), andcombination of sucrose with methionine (test #11).

TABLE 10 Purity of Fusion Protein 1 added with various excipients after4 days at 40° C. SEC-HPLC (%) #Test HHMW^(#) HMW^(a) Main peak^(b) LMW*Before treatment — 4.07 95.93 — 0 2.11 8.35 89.21 0.33 1 1.59 6.81 91.230.37 2 1.26 5.91 92.66 0.18 3 1.74 7.21 90.74 0.31 4 1.84 7.48 90.310.37 5 2.09 7.99 89.63 0.30 6 1.64 6.89 90.93 0.54 7 2.44 8.23 88.940.40 8 2.56 8.71 88.43 0.30 9 2.26 8.30 89.08 0.36 10 9.57 16.38 73.880.17 11 1.52 6.32 91.81 0.36 ^(a)MW: aggregate as high molecule weightspecies with retention time at 8.3 ± 0.1 min ^(b)Main peak retentiontime is 9.5 ± 0.1 min, which represents the non-aggregated dimeric formof Fusion Protein 1. ^(#)HHMW: aggregate as higher molecule weightspecies which refer to a peak that has retention time at 7.6 ± 0.1 min*LMW: low molecular weight with retention time at 13.5 ± 0.1 min “—”:Not detectable

Combinations of the remaining polyols with NaCl had slightly increased %of aggregate (Table 10, tests #6, 7, 8, 9) compared to tests withoutNaCl. In the combination of arginine and sucrose, the purity decreasedto 73.88% with over 16% aggregate after stored at accelerated condition(Table 10, test #10). Consequently, trehalose, sucrose, and mannitolwere selected to proceed to the design of experiment investigation.

Optimize Composition Candidate Formulations by DoE

To investigate the significance resulting from the stabilizer candidatesand clarify the cross-effect of NaCl with individual polyol, theselected polyols and various concentrations of NaCl were tested via theplanned DoE as shown in Table 11. The accelerated study conditions andanalysis are summarized in Table 12. According to the SEC-HPLC results(Table 13), 25 mM His buffer pH 6.0 with 200 mM polyol/sugar (especiallytrehalose and sucrose) provided the best protection of Fusion Protein 1against thermal stress to maintain a high percent purity of FusionProtein 1.

TABLE 11 DoE plan to test 80 mg/mL Fusion Protein 1 with variousconcentrations of ingredients. Test # Polyol* (mM) Sodium Chloride (mM)1 112.5 25 2 200 50 3 112.5 25 4 200 0 5 200 50 6 200 0 7 25 0 8 112.525 9 25 0 10 25 50 11 25 50 *polyol means trehalose, sucrose, ormannitol, each tested individually with or without NaCl.

TABLE 12 Accelerated condition testing. Storage Condition Time pointsAnalysis 40 ± 2° C./75 ± 5% RH Days 0, 4, and 7 Purity Osmolality 25 ±2° C./60 ± 5% RH Days 0 and 7 5 ± 3° C. Days 0 and 7

Following storage of 80 mg/mL Fusion Protein 1 samples at 40° C. for 4and 7 days (Table 12), in 25 mM histidine buffer at pH 6.0 formulatedwith 200 mM polyols stabilized Fusion Protein 1 against thermal stress,maintained the relative purity >80%, and provided the best resultingpurity of Fusion Protein 1 (Test #4 and 6 in Table 13). The samples withpolyol and 50 mM NaCl (tests #2 and #5) all showed lower purity thansamples with polyol alone (tests #4 and 6). This effect also wasobserved for samples with 25 mM polyol and no NaCl (tests #7 and 9)compared to samples with 25 mM polyol and 50 mM NaCl (tests #10 and 11).The differences between these combinations were not differentiated at 4°C. and 25° C. (data not shown).

TABLE 13 Purity and osmolality analysis of Fusion Protein 1 samplesstored at 40° C. ^(#)Relative Purity of Main Peak Analyzed by SEC-HPLC(%) Osmolality Sample 4 days 7 days (mOsm/kg) Trehalose Test 1 86.7078.32 256 Test 2 86.83 80.97 451 Test 3 86.49 79.29 247 Test 4 90.2385.48 312 Test 5 87.30 81.05 432 Test 6 90.31 85.41 304 Test 7 86.5079.85 77 Test 8 86.51 79.30 253 Test 9 86.51 79.83 73 Test 10 82.2272.47 195 Test 11 81.87 72.32 201 Mannitol Test 1 85.30 77.47 246 Test 285.92 78.27 415 Test 3 86.60 78.98 231 Test 4 89.01 83.62 300 Test 585.95 78.49 417 Test 6 89.41 83.93 295 Test 7 86.34 79.15 77 Test 885.49 77.51 239 Test 9 86.70 79.88 75 Test 10 81.22 71.36 193 Test 1181.42 71.74 186 Sucrose Test 1 86.92 79.71 256 Test 2 87.51 80.38 431Test 3 86.64 78.89 249 Test 4 90.10 85.21 301 Test 5 87.62 81.60 431Test 6 90.43 85.59 325 Test 7 86.88 79.64 77 Test 8 86.84 79.17 248 Test9 87.12 79.89 75 Test 10 82.13 72.52 194 Test 11 82.43 72.61 195^(#)Relative purity showed the purity of samples at 40° C. which wasnormalized with T0 samples (baseline).

Although higher histidine buffer with 200 mM polyol maintained qualityattributes of protein, osmolality must be maintained to the ocularphysiology range of 280-310 mOsm/kg. Therefore, 300 mOsm/kg was set asthe target DoE statistical calculation. The composition of ingredientsselected to formulate Fusion Protein 1 at 80 mg/mL were 25 mM histidinebuffer at pH 6.0, 190 mM trehalose, sucrose or combination of trehaloseand sucrose.

Assess Potential Excipients Composition of the Formula

To evaluate the compatibility of suitable ingredients in the combinedformula, further tests examined 80 mg/mL Fusion Protein 1 for use in 4candidate formulations. In this study, 0.03% polysorbate 20 (PS20) orpolysorbate 80 (PS80) were added together with trehalose or sucrose toassess Fusion Protein 1 attributes after treated with acceleratedcondition for a week and/or a month, various freeze-thaw cycles between−20° C. and room temperature (RT), and agitation after 24 and 48 hours(Tables 14A-14B). Short-term compatibility of the container was testedin parallel.

TABLE 14A Candidate formulations added with cryoprotectants and analyzedfor 28 days at 4° C. and 40° C. Time point Test Container AttributesCondition T0 D 4 D 7 D 14 D 28 Thermal 2R Type I Purity  4° C. baseline— — V V borosilicate Potency 40° C. V V — — glass vial Sub- visibleparticulate Integrity Content V = tested time point; T = time; D = Day.

TABLE 14B Candidate formulations added with cryoprotectants and testedin Freeze/thaw or Agitation Test Container Attributes TreatmentCycles/Time point Freeze- 0.2 mL Purity Freeze: at −20° C. for 23 hr 3cycles 6 cycles thaw cryovial Potency Thaw: at 25° C. for 1 hr Agitation2R Type I Sub-visible Shaking: 220 rpm at 25° C. 24 hr 48 hrborosilicate particulate glass vial Integrity Blank control: stationaryContent at 25° C.

The 4 candidate formulation compositions showed similar observationsabout the changes of attributes for the thermal stress studies. FusionProtein 1 at 80 mg/mL concentration was sensitive to the temperature at40° C., which caused main peak reduction to less than 95% and over 5%aggregate by 7 days. This was not observed at 4° C. storage for at leastone month (Tables 15 and 16). Moreover, the relative potency of VEGFand/or integrin αvβ3 binding compared to reference was within targetrange (70-130%) and showed slight changes on VEGF binding and no obviouschanges of αvβ3 binding following storage at accelerated conditions.

In particular, size exclusion chromatography studies indicated that aformulation of 25 mM histidine, 190 mM trehalose, and 0.03% PS20, pH 6.0enabled better stability of Fusion Protein 1 than other formulations atboth 4° C. and 40° C.

TABLE 15 Stability results of the thermal test at 4° C. Relative TimeTested SEC-HPLC (%) Potency* (%) point Conc. Main VEGF αvβ3 CandidateComponent (days) (mg/mL) HMW^(a) Peak^(b) binding binding Reference 25mM histidine, NA 37.7 NA NA 100 100 1# 6% sucrose, 20 mM NaCl, 0.03%PS20, pH 6.0 1 25 mM histidine, 0 73.5 0.94 99.06 86.2 96.9 190 mM 1473.1 1.31 98.69 87.7 91.8 trehalose, 28 74.5 1.78 98.22 88.3 119.7 0.03%PS20 2 25 mM histidine, 0 78.7 0.83 99.17 93.3 85.9 190 mM sucrose, 1483.5 1.47 98.53 90.9 96.4 0.03% PS20 28 85.8 1.99 98.01 87.8 131.5 3 25mM histidine, 0 81.9 0.85 99.15 86.6 141.8 190 mM 14 83.0 1.46 98.5488.6 107.3 trehalose, 28 85.4 2.04 97.96 91.3 134.9 0.03% PS80 4 25 mMhistidine, 0 80.6 0.74 99.26 90.0 118.5 190 mM sucrose 14 81.6 1.4198.59 95.6 109.9 0.03% PS80 28 81.9 1.99 98.02 89.7 114.7 ^(a)HMW:aggregate as high molecule weight species with retention time at 8.3 ±0.1 min #Reference 1 formulated in 25 mM histidine, 6% sucrose, 20 mMNaCl, 0.03% PS20, pH 6.0 ^(b)Main peak retention time is 9.5 ± 0.1 min,which represents non-aggregated dimeric form of Fusion Protein 1*relative potency was planned to be within 70-130% compared toreference. NA, not applicable.

TABLE 16 Stability results of the thermal test at 40° C. Relative TimePotency* (%) point Conc. SEC-HPLC (%) VEGF αvβ3 Candidate Component(days) (mg/mL) HMW^(a) Main^(b) binding binding Reference 25 mM NA 37.7NA NA 100 100 1^(#) histidine, 6% sucrose, 20 mM NaCl, 0.03% PS20, pH6.0 1 25 mM 0 73.5 0.94 99.06 86.2 96.9 histidine, 190 4 74.2 4.18 95.8280.8 122.5 mM trehalose, 7 72.9 6.43 93.58 82.1 95.5 0.03% PS20 2 25 mM0 78.7 0.83 99.17 93.3 85.9 histidine, 190 4 82.6 5.21 94.79 83.8 92.7mM sucrose, 7 79.5 7.24 92.76 87.7 94.1 0.03% PS20 3 25 mM 0 81.9 0.8599.15 86.6 141.8 histidine, 190 4 83.6 5.09 94.91 82.9 87.4 mMtrehalose, 7 82.6 7.34 92.66 78.2 102.7 0.03% PS80 4 25 mM 0 80.6 0.7499.26 90.0 118.5 histidine, 190 4 80.3 4.85 95.15 86.5 70.2 mM sucrose 781.7 7.38 92.63 77.7 112.7 0.03% PS80 ^(#)Reference 1 formulated in 25mM histidine, 6% sucrose, 20 mM NaCl, 0.03% PS20, pH 6.0 ^(a)HMW:aggregate as high molecule weight species with retention time at 8.3 ±0.1 min ^(b)Main peak retention time is 9.5 ± 0.1 min, which representsnon-aggregated dimeric form of Fusion Protein 1 *Target relative potencywas planned to be within 70-130% compared to reference. NA, notapplicable.

Fusion Protein 1 at 80 mg/mL formulated in the candidate formulation wastested for the quality attributes after freezing at −20° C. forapproximately 23 hours and thaw at 25° C. for at least 1 hour for 3 and6 cycles. Analysis by SEC-HPLC did not detect any obvious changes in thepercent main peak of Fusion Protein 1 and its aggregate (Table 17). Theremaining protein was more than 95% and the aggregate was less than 5%.Additionally, the potency of Fusion Protein 1 binding to VEGF orintegrin αvβ3 compared to the reference was decreased to less than 70%in candidates #3 and #4 (Table 17).

TABLE 17 Stress stability tests after 3 and 6 freeze-thaw (F/T) cycles.Relative No. of Conc. SEC-HPLC (%) Potency* (%) F/T (mg/mL) Main VEGFαvβ3 Candidate Composition cycle By A280 HMW^(a) Peak^(b) bindingbinding Reference 25 mM histidine, 4 37.7 NA NA 100 100 1# 6% sucrose,20 mM NaCl, 0.03% PS20, pH 6.0 1 25 mM histidine, 0 73.5 0.94 99.06 86.296.9 190 mM trehalose, 3 77.6 1.34 98.66 76.9 100.3 0.03% PS20 6 80.11.11 98.90 83.2 106.4 2 25 mM histidine, 0 78.7 0.83 99.17 93.3 85.9 190mM sucrose, 3 82.3 1.32 98.68 83.0 96.7 0.03% PS20 6 84.9 1.15 98.8593.2 153.0 3 25 mM histidine, 0 81.9 0.85 99.15 86.6 141.8 190 mMtrehalose, 3 85.5 1.35 98.65 77.0 100.7 0.03% PS80 6 86.8 0.99 99.0182.8 54.8* 4 25 mM histidine, 0 80.6 0.74 99.26 90.0 118.5 190 mMsucrose 3 87.4 1.32 98.68 57.5* 99.2 0.03% PS80 6 88.2 1.09 98.91 87.599.5 #Reference 1 formulated in 25 mM histidine, 6% sucrose, 20 mM NaCl,0.03% PS20, pH 6.0 ^(a)HMW: aggregate as high molecule weight specieswith retention time at 8.3 ± 0.1 min. ^(b)Main peak retention time is9.5 ± 0.1 min, which represents non-aggregated dimeric form of FusionProtein 1. *Target relative potency was planned to be within 70-130%compared to reference. NA, not applicable.

The agitation studies tested protein stability to mimic potentialhandling and transportation with Fusion Protein 1 in liquid form. Theconditions are accelerated by shaking the vials at 220 rpm for 24 and 48hours under a 25° C. environment. Treating the protein samples undersuch a rigorous condition did impact the protein purity and binding tothe major targets compared to samples exposed at the stationarycondition and baseline (Table 18).

TABLE 18 Stress stability test by agitation for 24 and 48 hours.Relative Tested SEC-HPLC (%) Potency* (%) Duration Conc. Main VEGF αvβ3Candidate Component Test (hrs) (mg/mL) HMW^(a) Peak^(b) binding bindingReference 25 mM histidine, NA NA 37.73 NA NA 100 100 1# 6% sucrose, 20mM NaCl, 0.03% PS20, pH 6.0 1 25 mM histidine, Baseline 0 73.5 0.9499.06 86.2 96.9 190 mM trehalose, Stationary 24 72.5 1.30 98.7 81.3105.5 0.03% PS20 Agitation 72.1 1.44 98.56 82.3 118.7 Stationary 48 71.51.74 98.26 103.4 118.7 Agitation 77.8 1.53 98.47 76.3 111.0 2 25 mMhistidine, Baseline 0 78.7 0.83 99.17 93.3 85.9 190 mM trehalose,Stationary 24 83.8 1.43 98.57 79.1 98.6 0.03% PS20 Agitation 87.7 1.5898.42 76.5 89.3 Stationary 48 82.9 1.86 98.14 108.0 119.0 Agitation 85.31.79 98.21 84.5 78.7 3 25 mM histidine, Baseline 0 81.9 0.85 99.15 86.6141.8 190 mM trehalose, Stationary 24 83.1 1.50 98.5 75.8 88.9 0.03%PS80 Agitation 83.3 1.61 98.39 78.8 103.0 Stationary 48 85.9 1.88 98.1297.7 103.3 Agitation 82.3 1.72 98.28 81.5 94.2 4 25 mM histidine,Baseline 0 80.6 0.74 99.26 90.0 118.5 190 mM trehalose, Stationary 2481.3 1.50 98.5 80.6 131.8 0.03% PS80 Agitation 80.3 1.58 98.42 82.7123.4 Stationary 48 86.4 1.89 98.11 105.4 183.1 Agitation 78.5 1.7198.29 85.7 134.4 #Reference 1 formulated in 25 mM histidine, 6% sucrose,20 mM NaCl, 0.03% PS20, pH 6.0 ^(a)HMW: aggregate as high moleculeweight species with retention time at 8.3 ± 0.1 min. ^(b)Main peakretention time is 9.5 ± 0.1 min, which represents non-aggregated dimericform of Fusion Protein 1. *Target relative potency was planned to bewithin 70-130% compared to reference. NA, not applicable.

In addition to the critical quality attributes such as quantity, purity,and potency, the formation and distribution of sub-visible particlesafter the stress conditions were examined. According to dynamic lightscattering (DLS) analysis, the observed sub-visible particles werecategorized by size, % polydispersity, and the proportion of differentsize of particles. The results indicated that the progress to obtainconcentrated Fusion Protein 1 to the desired concentration 80 mg/mL wasnot monitored appropriately, thus, not only the major Fusion Protein 1molecule with radius approximately 5 nm was detected, but also theaggregate can be seen as larger size particles i.e., 10-100 nm or100-1000 nm at baseline (DO). It was not possible to conclude whichcandidate formulation buffer worked the best by DLS.

Moreover, non-reducing and reducing SDS-PAGE analyses were not able todifferentiate the change of integrity of Fusion Protein 1 after treatingwith stress conditions. Osmolality of these tested candidateformulations (Table 19) showed that except for candidate #4 which showedlower osmolality, the other formulations were of similar range.

TABLE 19 Osmolality of candidate formulations. Osmolality FormulationComposition (mOsm/kg) 1 25 mM Histidine, 190 mM trehalose, 248 0.03%PS20, pH 6.0 2 25 mM Histidine, 190 mM Sucrose, 257 0.03% PS20, pH 6.0 325 mM Histidine, 190 mM trehalose, 250 0.03% PS80, pH 6.0 4 25 mMHistidine, 190 mM Sucrose, 209 0.03% PS80, pH 6.0

Conclusion

Based on the results, composition of formulation 1 with 25 mM His buffercontaining 190 mM trehalose and 0.03% PS20 pH 6.0, was nominated to betested in a pilot drug product for a 12 month stability study.

Example 4. Formulation of Fusion Protein 1 for Intravitreal Injection

A stability study was conducted to develop the final formulation ofFusion Protein 1.

Bulk Drug Substance Concentration Testing

To select the suitable concentration to support final bulk drugsubstance of Fusion Protein 1 through the manufacturing process, both 40and 80 mg/mL Fusion Protein 1 were formulated in the leading formulationcomponent (25 mM histidine, 190 mM trehalose, and 0.03% PS20, pH 6.0).Formulations with both concentrations of Fusion Protein 1 were testedunder short-term accelerated condition to assess critical qualityattributes for any changes by SEC-HPLC, DLS, and SDS-PAGE analyses.

Protein quantity exhibited variably among the 40 mg/mL samples and 80mg/mL samples for short term storage at 40° C. for 7 days (Table 20). Aslightly greater amount of aggregate was observed at 80 mg/mL. Resultsfor samples stored at 40° C., indicated that Fusion Protein 1 formedless high molecule weight aggregate at 40 mg/mL compared to 80 mg/mL byday 4 and day 7. The test #2 protein sample at 40 mg/mL with NaClexhibited >5% aggregate detected. Freeze/Thaw cycle tests did not showobvious changes of the aggregate % or purity for both 40 and 80 mg/mLsamples (data not shown).

TABLE 20 Stability of Fusion Protein 1 at 40 and 80 mg/mL in formulationbuffer with and without NaCl at 40° C. Tested SEC-HPLC (%) DurationConc. Main Sample Composition (days) (mg/mL) HMW^(a) Peak^(b) Test 1 40mg/mL Fusion 0 42.8 1.52 98.48 Protein 1 4 48.3 2.83 97.17 25 mMHistidine, 49.9 2.93 97.07 190 mM trehalose, 7 42.2 3.69 96.31 0.03%PS20, pH 6.0 43.3 3.74 96.26 Test 2 40 mg/mL Fusion 0 42.9 1.63 98.38Protein 1 4 45.4 4.22 95.78 25 mM Histidine, 46.3 4.26 95.74 190 mMtrehalose, 7 42.5 5.20 94.02 10 mM NaCl, 42.4 5.21 94.07 0.03% PS20, pH6.0 Test 3 80 mg/mL Fusion 0 87.3 1.63 98.37 Protein 1 4 90.8 4.75 94.6725 mM Histidine, 90.0 4.73 94.64 190 mM trehalose, 7 86.2 6.55 92.340.03% PS20, pH 6.0 86.2 6.52 92.49 Test 4 80 mg/mL Fusion 0 85.6 1.6698.34 Protein 1 4 88.1 5.08 94.22 25 mM Histidine, 92.9 5.08 94.22 190mM trehalose, 7 84.3 6.95 91.78 10 mM NaCl, 84.4 6.98 91.90 0.03% PS20,pH 6.0 ^(a)HMW: aggregate as high molecule weight species with retentiontime at 8.3 ± 0.1 min. ^(b)Main peak retention time is 9.5 ± 0.1 min,which represents non-aggregated dimeric form of Fusion Protein 1.

DLS was used to analyze sub-visible particles and distribution and allprotein samples contained particles in size ranges from 10 nm-100 nmand/or 100-1000 nm at baseline, which made it impossible todifferentiate changes after stress tests.

Pilot Drug Product Stability Test

Based on the studies described above, and where Fusion Protein 1maintained the most consistent purity based on SEC-HPLC, the selectedformulation composition for Fusion Protein 1 was as follows: 40 mg/mLFusion Protein 1, 25 mM histidine, 190 mM trehalose and 0.03% PS20 at pH6.0. To verify and evaluate the compatibility with the container systemas well as to collect initial stability, 40 mg/mL Fusion Protein 1 wasprepared in this formula, filled in borosilicate Type I glass vials(rubber stoppered and flip-cap sealed), and incubated under threeconditions, −25° C. to −15° C., 2-8° C., and 25° C./60% relativehumidity (RH) (Table 21).

TABLE 21 Formulation development study plan. Test Time Point Condition T= 0 2 wks 1 M 3 M 6 M 9 M 12 M 25° C./60% RH X X X X X — — 4° C. X X X XX X −20° C. in 2R vial X X X X — — X = time point to check the qualityattributes of the protein. Wks = weeks; M = months; T = time

The results of the study indicated that 40 mg/mL Fusion Protein 1 wasstable when stored at −20° C. for at least 12 months (Table 22). After 6months in the storage condition at 4° C., there was a slight decrease inpurity and increase of aggregate (Table 23). In terms of acceleratedstorage at 25° C., SEC-HPLC detected increased aggregate % through 6months, however the main peak remained ≥95% and the molecule specificpotency and integrity (i.e., data of capillary electrophoresis sodiumdodecyl sulfate (CE-SDS)) were maintained with minimal changes (Table24).

TABLE 22 Pilot drug product stability of Fusion Protein 1 stored at −20°C. VEGF binding CE-SDS Time Conc. SEC-HPLC (%) Relative Non- Test point(mg/mL) Main Potency^(@) reducing Reducing Article (months) By A280HMW^(a) Peak^(b) %/EC₅₀ (%) (%) Z19003^(#) 0 41 0.8 99.20 92/0.408 10099.3 1 41.6 0.94 99.06 96/0.368 100 100 3 42.3 1.14 98.86 98/0.393 10099.5 6 41.0 0.72 99.28 97/0.397 100 99.3 9 42.2 0.95 99.05 95/0.407 10099.6 12 43.4 0.71 99.29 93/0.370 100 99.4 Z19003^(#) 0 41 0.87 99.1392/0.408 100 99.3 (1x F/T 1 41.8 0.87 99.13 95/0.374 100 99.3 cycle) 342.3 0.93 99.07 102/0.380  100 99.1 6 42.0 0.76 99.24 96/0.401 100 98.89 41.7 0.99 99.01 95/0.409 100 99.5 12 42.3 0.72 99.28 93/0.372 100 99.5^(a)HMW: aggregate as high molecule weight species with retention timeat 8.3 ± 0.1 min. ^(b)Main peak retention time is 9.5 ± 0.1 min, whichrepresents non-aggregated dimeric form of Fusion Protein 1. ^(@)Relativepotency is calculated based on EC50 value compared to the baseline (D0). ^(#)Z19003 is Lot No. of the test article.

TABLE 23 Pilot drug product stability result of Fusion Protein 1 at 4°C. VEGF binding CE-SDS Conc. SEC-HPLC Relative Non- Test Timepoint(mg/mL) Main Potency^(@) reducing Reducing Article (months) By A280HMW^(a) Peak^(b) %/EC₅₀ (%) (%) Z19003^(#) 0 41.0 0.8 99.20 92/0.408 10099.3 0.5 41.7 0.97 99.04 98/0.250 100 99.5 1 42.4 1.15 98.85 93/0.381100 99.8 3 41.8 1.60 98.41 93/0.461 100 99.2 6 41.0 1.41 98.59 89/0.431100 99.4 9 41.3 2.18 97.83 101/0.377  100 99.4 12 42.0 1.80 98.2095/0.365 100 99.4 Z19003^(#) 0 41.0 0.87 99.13 92/0.408 100 99.3 (1x F/T0.5 42.1 0.93 99.07 97/0.254 100 99.2 cycle) 1 42.3 1.12 98.88 91/0.389100 99.4 3 44.7 1.47 98.53 86/0.499 100 99.4 6 41.6 1.43 98.57 89/0.432100 99.3 9 41.5 2.21 97.79 95/0.398 100 99.6 12 41.4 1.94 98.06 97/0.357100 99.4 ^(a)HMW: aggregate as high molecule weight species withretention time at 8.3 ± 0.1 min. ^(b)Main peak retention time is 9.5 ±0.1 min, which represents non-aggregated dimeric form of FusionProtein 1. ^(@)Relative potency is calculated based on EC₅₀ valuecompared to the baseline (D 0). ^(#)Z19003 is Lot No. of the testarticle.

TABLE 24 Pilot drug product stability result of Fusion Protein 1 at 25°C. VEGF binding CE-SDS Conc. SEC-HPLC Relative Non- Test Timepoint(mg/mL) Main Potency^(@) reducing Reducing Article (months) By A280HHMW^(#) HMW^(a) Peak^(b) %/EC₅₀ (%) (%) Z19003^(#) 0 41.0 — 0.8 99.2092/0.408 100 99.3 0.5 42.9 — 1.40 98.60 94/0.263 100 99.3 1 42.6 — 1.8598.15 99/0.370 100 99.1 3 42.8 0.365 3.21 96.43 92/0.421 100 98.7 6 41.8— 3.30 96.70 78/0.494 100 98.7 Z19003^(#) 0 41.0 — 0.87 99.13 92/0.408100 99.3 (1x F/T 0.5 43.8 — 1.42 98.59 91/0.271 100 99.2 cycle) 1 40.9 —1.89 98.11 95/0.373 100 99.3 3 44.5 0.214 2.90 96.88 91/0.424 100 98.7 641.3 — 3.49 96.51 80/0.480 100 86.8 ^(a)HMW: aggregate as high moleculeweight species with retention time at 8.3 ± 0.1 min. ^(b)Main peakretention time is 9.5 ± 0.1 min, which represents non-aggregated dimericform of Fusion Protein 1. ^(#)HHMW: aggregate as higher molecule weightspecies which refer to a peak that has retention time at 7.6 ± 0.1 min^(@)Relative potency is calculated based on EC₅₀ value compared to thebaseline (D 0). ^(#)Z19003 is Lot No. of the test article.

According to the DLS analysis, sub-visible particles were not detectedwhen the Test Articles were stored at −20° C. and 4° C. for at least 12months, except for one sample stored at 4° C. which experienced onefreeze/thaw cycle at the 12 month time point and formed larger sizeparticles (radius 2208 nm) in a trace amount (2%). Larger size particles(radius 2195 and 2264 nm) were detected in 3 and 6-months samples storedat 25° C.

A backup of the sample stored at 4° C. was analyzed to confirm thepresence of sub-visible particles observed at 12 months. This sample wasalso stored for 12 months and underwent a freeze/thaw cycle. Sub-visibleparticles were observed in the studied backup sample. Therefore, FusionProtein 1 is sensitive to the freeze/thaw stress following storage for12 months at 4° C. Consequently, without freeze/thaw cycle, FusionProtein 1 can be stored at 4° C. for at least 12 months. However, ifFusion Protein 1 is to be stored longer than 6 months then storage at−20° C. is recommended.

The viscosity measurement of the formulation was obtained using aviscometer. The subject formulation (40 mg/mL Fusion Protein 1 in 25 mMhistidine buffer solution with selected ingredients) had low viscositywith 5.402 cP (centipoise) (Table 25). The selected formulation hasfavorable thermal properties for the protein, as shown by therepresentative differential scanning calorimetry (DSC) thermogram inFIG. 9 . The deconvoluted thermogram has 4 thermal transition peaks,with Tm values at 61.47° C., 66.51° C., 81.55° C., to 84.21° C.

TABLE 25 Viscosity of formulation solution and Fusion Protein 1 in thesolution at 4° C. Test Samples Formulation solution Protein Lot No.Z19003 (Fusion Protein 1, (Fusion Protein 1, 0 mg/mL 25 mM 40 mg/mL, 25mM histidine, 190 mM histidine, 190 mM trehalose, 0.03% trehalose, 0.03%polysorbate 20, polysorbate 20, pH 6.0) pH 6.0) Mean (n = 3 or 4)* 4.495cP 5.402 cP Standard deviation 0.274 0.884 *Mean value captured fromthree or four individual test results.

Conclusion

In light of the results of this study, Fusion Protein 1 formulated at 40mg/mL in 25 mM histidine, 190 mM trehalose and 0.03% PS20 pH 6.0 wascompatible with the selected container closure system and remainedstable for at least 12 months when stored at −20° C. and 2° C.-8° C.

Example 5. Long-Term Stability Study of Fusion Protein 1

A study was conducted to examine the long-term stability of FusionProtein 1 formulated at 40 mg/mL in 25 mM histidine, 190 mM trehaloseand 0.03% PS20, pH 6.0, at various temperatures. Fusion Protein 1exhibited stability over two years following storage at −70° C. (Table26). The variation of EC₅₀ was observed in αvβ3 and α5β1 binding atmonths 9, 18, and 24 (T9, T18, and T24) and months 9, 12, and 24 (T9,T12, and T24). This variation was investigated and indicated that thismight be due to expired testing reagents.

TABLE 26 Summary of Stability Data of Fusion Protein 1 at −70° C. ± 10°C. Test Items T0 T1 T3 T6 T9 T12 T18 T24 Appearance BY6 BY6 BY6 BY6 BY6BY6 BY5 BY5 pH 6.2 6.2 6.2 6.2 6.1 6.1 6.1 6.1 A280 (mg/mL) 41 41 41 4140 39 41 40 SEC-HPLC/UV, 98.8 99.2 99.1 99.2 99.1 99.2 99.1 99.2 mainpeak (%) SEC-HPLC/UV, 1.2 0.8 0.9 0.8 0.9 0.8 0.9 0.8 aggregate (%)Non-reducing 100 100 100 100 100 100 100 100 CE-SDS (%) Reducing 99.399.3 99.4 99.4 99.3 99.3 99.3 99.3 CE-SDS (%) Capillary 99.78 99.6899.82 99.78 99.82 99.66 99.55 99.74 isoelectric focusing (cIEF) (%)VEGF-A₁₆₅ 0.408 0.370 0.350 0.478 0.385 0.404 0.427 0.489 ELISA bindingaffinity (EC₅₀) αvβ3 ELISA 0.012 0.014 0.015 0.014 0.032 0.006 0.0230.085 binding affinity (EC₅₀) α5β1 ELISA 0.251 0.086 0.088 0.090 0.1800.031 0.047 0.112 binding affinity (EC₅₀) T = monthly stability storageof material BY = brownish-yellow *α5β1 binding ELISA was furtheroptimized after 0 M, T1 data was used for the trend analysis of α5β1binding ELISA stability.

A study examining the stability of Fusion Protein 1 formulated at 40mg/mL in 25 mM histidine, 190 mM trehalose and 0.03% PS20, pH 6,following storage at −20° C. over 36 months is ongoing. A summary of thestability results following storage at 9 months at −20° C. is shown inTable 27.

TABLE 27 Stability of Fusion Protein 1 at −20° C. ± 5° C., Upright TestItems T0 T1 T3 T6 T9 T12 T18 T24 T36 Appearance BY6, BY5, BY5, BY6,Between BY5 TBD TBD TBD TBD essentially essentially essentiallyessentially and BY6, free from free from free from free from essentiallyvisible visible visible visible free from particulates particulatesparticulates particulates visible particulates pH 6.1 6.1 6.1 6.1 6.1TBD TBD TBD TBD Osmolality 268 267 267 255 261.7* TBD TBD TBD TBD(Osmo/kg) cIEF (%) 99.97) 99.89 99.92 99.78 99.85 TBD TBD TBD TBD A280(mg/mL) 38 39 38 37 37 TBD TBD TBD TBD SEC-HPLC/UV, 99.2 99.2 97.8 99.299.1 TBD TBD TBD TBD main peak (%) SEC-HPLC/UV, 0.8 0.8 2.2 0.8 0.9 TBDTBD TBD TBD aggregate (%) Non-reducing 100 100 100 100 100 TBD TBD TBDTBD CE-SDS (%) Reducing 99.3 99.3 99.4 99.4 99.2 TBD TBD TBD TBD CE-SDS(%) VEGF-A₁₆₅ binding 109 99 104 96 106 TBD TBD TBD TBD relative potency(%) αvβ3 binding relative 129 114 125 88 101 TBD TBD TBD TBD potency (%)α5β1 binding relative 129 100 112 113 104 TBD TBD TBD TBD potency (%)Sub-visible 10 μm 1 NT NT 17 NT TBD NT TBD TBD particulate 25 μm 0 NT NT0 NT TBD NT TBD TBD matter 50 μm 0 NT NT 0 NT TBD NT TBD TBD T = monthlystability storage of material; BY = brownish-yellow; NT = not tested;TBD = to be determined; TO = Test omitted; *data collected fromosmolality investigation

Additionally, a study examining the stability of Fusion Protein 1formulated at 40 mg/mL in 25 mM histidine, 190 mM trehalose and 0.03%PS20, pH 6, following storage at 5° C. over 36 months is ongoing. Asummary of the stability results following storage at 9 months at 5° C.either upright or inverted is shown in Table 28 and Table 29,respectively.

TABLE 28 Stability of Fusion Protein 1 at 5° C. ± 3° C., Upright TestItems T0 T1 T3 T6 T9 T12 T18 T24 T36 Appearance BY6, BY5, BY5, BY6,Between TBD TBD TBD TBD essentially essentially essentially essentiallyBY5 and free from free from free from free from BY6, visible visiblevisible visible essentially particulates particulates particulatesparticulates free from visible particulates pH 6.1 6.1 6.1 6.1 6.1 TBDTBD TBD TBD Osmolality 268 267 267 267 266 TBD TBD TBD TBD (mOsmo/kg)cIEF (%) 99.97 99.93 99.78 99.89 99.92 TBD TBD TBD TBD A280 (mg/mL) 3838 38 38 38 TBD TBD TBD TBD SEC-HPLC/UV, 99.2 99.0 99.3 98.7 98.5 TBDTBD TBD TBD main peak (%) SEC-HPLC/UV, 0.8 1.0 0.7 1.3 1.5 TBD TBD TBDTBD aggregate (%) Non-reducing 100 100 100 100 100 TBD TBD TBD TBDCE-SDS, (%) Reducing 99.3 99.3 99.3 99.3 99.1 TBD TBD TBD TBD CE-SDS,(%) VEGF-A₁₆₅ 109 108 102 102 102 TBD TBD TBD TBD binding relativepotency (%) αvβ3 binding 129 89 100 104 95 TBD TBD TBD TBD relativepotency (%) α5β1 binding 129 103 103 105 102 TBD TBD TBD TBD relativepotency (%) T = monthly stability storage of material; BY =brownish-yellow; TBD = to be determined.

TABLE 29 Stability of Fusion Protein 1 at 5° C. ± 3° C., Inverted TestItems T0 T1 T3 T6 T9 T12 T18 T24 T36 Appearance BY6, BY5, BY5, BY6,Between TBD TBD TBD TBD essentially essentially essentially essentiallyBY5 and free from free from free from free from BY6, visible visiblevisible visible essentially particulates particulates particulatesparticulates free from visible particulates pH 6.1 6.1 6.1 6.1 6.1 TBDTBD TBD TBD Osmolality 268 268 267 267 267 TBD TBD TBD TBD (mOsmo/kg)cIEF (%) 99.97 99.89 99.80 99.85 99.98 TBD TBD TBD TBD A280 (mg/mL) 3838 38 38 38 TBD TBD TBD TBD SEC-HPLC/UV, 99.2 99.0 98.8 98.6 98.5 TBDTBD TBD TBD main peak (%) SEC-HPLC/UV, 0.8 1.0 1.2 1.4 1.5 TBD TBD TBDTBD aggregate (%) Non-reducing 100 100 100 100 100 TBD TBD TBD TBDCE-SDS (%) Reducing 99.3 99.4 99.3 99.3 99.2 TBD TBD TBD TBD CE-SDS (%)VEGF-A₁₆₅ 109 103 103 92 92 TBD TBD TBD TBD binding relative potency (%)αvβ3 binding 129 122 100 90 107 TBD TBD TBD TBD relative potency (%)α5β1 binding 129 101 127 94 99 TBD TBD TBD TBD relative potency (%)Sub-visible 10 μm 1 NT NT 23 NT TBD NT TBD TBD particulate 25 μm 0 NT NT4 NT TBD NT TBD TBD matter 50 μm 0 NT NT 0 NT TBD NT TBD TBD T = monthlystability storage of material; BY = brownish-yellow; NT = not tested;TBD = to be determined.

Furthermore, a study was performed that examined the stability of FusionProtein 1 formulated at 40 mg/mL in 25 mM histidine, 190 mM trehaloseand 0.03% PS20, pH 6, following storage at 25° C. over 6 months. Asummary of the stability results following storage at 6 months at 25° C.is shown in Table 30.

TABLE 30 Stability of Fusion Protein 1 at 25° C. ± 2° C./60% ± 5% RH,Upright Test Items T0 T1 T3 T6 Appearance BY6, BY5, BY5, BY5,essentially essentially essentially essentially free from free from freefrom free from visible visible visible visible particulates particulatesparticulates particulates pH 6.1 6.1 6.1 6.1 Osmolality 268 268 269 271(mOsm/kg) cIEF (%) 99.97 99.91 99.85 99.83 A280 (mg/mL) 38 38 38 38SEC-HPLC/UV, main 99.2 98.3 98.8 96.9 peak (%) SEC-HPLC/UV 0.8 1.7 1.23.1 aggregate (%) Non-reducing CE- 100 100 100 100 SDS (%) ReducingCE-SDS 99.3 99.4 98.9 98.5 (%) VEGF-A₁₆₅ binding 109 103 98 89 relativepotency (%) αvβ3 binding relative 129 116 125 111 potency (%) α5β1binding ELISA 129 108 126 89 (%) Sub- 10 μm 1 NT NT 18 visible 25 μm 0NT NT 2 particulate 50 μm 0 NT NT 2 matter T = monthly stability storageof material; BY, brownish-yellow; NT = not tested.

Example 6. Vascular Leakage Score in Rabbits Treated with theFormulation of Fusion Protein 1 Following Stimulation with VEGF-A₁₆₅

An in vivo study was conducted to test the efficacy of various doses ofFusion Protein 1 formulated in the above-described formulation ofinterest 9 (40 mg/mL of Fusion Protein 1 in 25 mM histidine, 190 mMtrehalose and 0.03% PS20, pH 6). Human VEGF-induced retinal vascularleakage was examined in Dutch Belted rabbits by defined leakage scorebased on standardized fluorescence angiography (FA) scoring images.Leakage score was categorized from 0 to 4 (0, major vessel verystraight, with some tortuosity of small vessels; 1, increased tortuosityof major vessels and/or vessel dilation; 2, leakage between majorvessels; 3, leakage between major and minor vessels, minor vessel stillvisible; 4, leakage between major and minor vessels, minor vessel notvisible).

Each rabbit received bilateral single intravitreal injection with 50 μL,each group had a total of 3 rabbits and 6 eyes. On Day 0 Rabbits wereadministered 50 μL of vehicle control, Avastin® (1.25 mg/eye), Eylea®(0.625 mg/eye), and increasing concentrations of Fusion Protein 1 (i.e.,0.03 mg, 0.1 mg, 0.3 mg, and 1 mg per eye). The rabbits were thenstimulated with 1000 ng of human VEGF-A₁₆₅ on Day 2 and FA was used toassess leakage score on Day 5.

Rabbits treated with as low as 0.03 mg of Fusion Protein 1 exhibited adecreased vascular leakage score (FIG. 10 ). Consequently, a dose as lowas 0.03 mg of Fusion Protein 1 effectively reduces vascular leakage.

Fusion Protein 1 doses were comparable with Avastin® and Eylea® in itsability to inhibit VEGF-induced retinal leakage. These data supportFusion Protein 1 to be suitable in treating angiogenic retinal diseasessuch as DME, nAMD, and RVO.

We claim:
 1. A pharmaceutical formulation, the formulation comprising:a) a fusion protein in a concentration of about 0.5 mg/mL to about 120mg/mL, b) a polyol or alcohol selected from a group consisting ofsucrose, trehalose, mannitol, sorbitol, benzyl alcohol, polyvinylalcohol, polyethylene glycol (PEG) 400-12000, in a concentration ofabout 1% to about 10% w/v, c) a buffering agent selected from a groupconsisting of sodium phosphate, histidine, sodium citrate, sodiumacetate, sodium bicarbonate, and trisodium citrate dihydrate in aconcentration of about 10 mM to about 50 mM, and d) a surfactant in aconcentration of about 0.01 to about 4% w/v, wherein the formulation isat a pH of about 5.5-7.5 and optionally, the formulation furthercomprises a polysaccharide selected from the group consisting of sodiumcarboxymethylcellulose, microcrystalline cellulose, or sodiumhyaluronate.
 2. The pharmaceutical formulation of claim 1, wherein thesurfactant is selected from a group consisting of polysorbate 20,polysorbate 80, and poloxamer
 188. 3. The pharmaceutical formulation ofclaim 2, wherein the surfactant is polysorbate
 20. 4. The pharmaceuticalformulation of claim 1, wherein the surfactant is in a concentration ofabout 0.03% w/v.
 5. The pharmaceutical formulation of claim 1, whereinthe fusion protein is in a concentration of about 1 mg/mL to about 90mg/mL.
 6. The pharmaceutical formulation of claim 5, wherein the fusionprotein is in a concentration of about 40 mg/mL.
 7. The pharmaceuticalformulation of claim 1, wherein the polyol is trehalose in aconcentration of about 25 mM to about 250 mM.
 8. The pharmaceuticalformulation of claim 8, wherein the polyol is trehalose in aconcentration of about 190 mM.
 9. The pharmaceutical formulation ofclaim 1, wherein the buffering agent is histidine in a concentration ofabout 10 mM to about 40 mM.
 10. The pharmaceutical formulation of claim9, wherein the buffering agent is histidine in a concentration of about25 mM.
 11. The pharmaceutical formulation of claim 1, wherein the fusionprotein comprises, from N-terminus to C-terminus in the following order:a) an extracellular domain of a Vascular Endothelial Growth Factorreceptor (VEGFR); b) an Fc domain of human immunoglobulin G; and c) anintegrin binding protein or its fragment thereof.
 12. The pharmaceuticalformulation of claim 11, wherein the fusion protein comprises SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO: 17, or SEQ ID NO:
 18. 13. Thepharmaceutical formulation of claim 1, wherein the pH is about 5.5 toabout 7.0.
 14. The pharmaceutical formulation of claim 13, wherein thepH is about 6.0.
 15. The pharmaceutical formulation of claim 1, whereinthe formulation is stable at −70° C., −20° C. and/or 5° C. for at least24 months.
 16. The pharmaceutical formulation of claim 1, wherein theformulation retains protein purity and potency after at least 6 monthsat −70° C., −20° C., 2-8° C., and/or 25° C.
 17. The pharmaceuticalformulation of claim 16, wherein the formulation retains protein purityand potency after at least 6 months at 2-8° C.
 18. The pharmaceuticalformulation of claim 1, wherein the formulation further comprises a saltin a concentration of about 10 mM to 50 mM.
 19. The pharmaceuticalformulation of claim 1, wherein the formulation further comprises atleast one amino acid in a concentration of about 10 mM to 50 mM.
 20. Thepharmaceutical formulation of claim 18, wherein the salt is selectedfrom sodium chloride, magnesium chloride, calcium chloride, or potassiumchloride.
 21. The pharmaceutical formulation of claim 19, wherein theamino acid is selected from the group consisting of arginine,methionine, proline, histidine, cysteine, lysine, glycine, aspartate,tryptophan, glutamate, and isoleucine.
 22. A pharmaceutical formulation,the formulation comprising: a) a fusion protein in a concentration of 40mg/mL, b) 25 mM histidine, c) 190 mM trehalose, sucrose, or mannitol, d)0.03% polysorbate 20 or polysorbate 80, wherein the formulation is at apH of about 6.0.
 23. The pharmaceutical formulation of claim 22, whereinthe fusion protein comprises, from N-terminus to C-terminus in thefollowing order: a) an extracellular domain of a Vascular EndothelialGrowth Factor receptor (VEGFR); b) an Fc Domain of human immunoglobulinG; c) an integrin binding protein or its fragment thereof.
 24. Thepharmaceutical formulation of claim 23, wherein the fusion proteincomprises SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO: 17, or SEQ ID NO: 18.25. The pharmaceutical formulation of claim 22, wherein the formulationis stable at −70° C., −20° C. and/or 5° C. for at least 24 months. 26.The pharmaceutical formulation of claim 22, wherein the formulationretains protein purity and potency after at least 6 months at −70° C.,−20° C., and/or 2-8° C.
 27. The pharmaceutical formulation of claim 22,wherein the formulation further comprises a salt in a concentration ofabout 10 mM to 50 mM.
 28. The pharmaceutical formulation of claim 22,wherein the formulation further comprises at least one amino acid in aconcentration of about 10 mM to 50 mM.
 29. The pharmaceuticalformulation of claim 27, wherein the salt is selected from sodiumchloride, magnesium chloride, calcium chloride, or potassium chloride.30. The pharmaceutical formulation of claim 28, wherein the amino acidis selected from the group consisting of arginine, methionine, proline,histidine, cysteine, lysine, glycine, aspartate, tryptophan, glutamate,and isoleucine.